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Textbook of Cosmetic Dermatology

Series in Cosmetic and Laser Therapy

Series Editors
Nicholas J. Gary P. Lask, and David J. Goldberg

Robert Baran and Howard Maibach, Textbook of Cosmetic Dermatology,

Fifth Edition, ISBN 9781482223934
Philippe Deprez, Textbook of Chemical Peels, Second Edition: Superficial,
Medium, and Deep Peels in Cosmetic Practice, ISBN 9781482223934
Jenny Kim, Gary Lask, and Andrew Nelson, Comprehensive Aesthetic
Rejuvenation: A Regional Approach, ISBN 9780415458948
David J. Goldberg and Alexander L. Berlin, Disorders of Fat and Cellulite:
Advances in Diagnosis and Treatment, ISBN 9780415477000
Neil S. Sadick, Paul J. Carniol, Deborshi Roy, and Luitgard Wiest, Illustrated
Manual of Injectable Fillers: A Technical Guide to the Volumetric Approach to
Whole Body Rejuvenation, ISBN 9780415476447
Kenneth Beer, Mary P. Lupo, and Vic A. Narurkar, Cosmetic Bootcamp
Primer: Comprehensive Aesthetic Management, ISBN 9781841846989
Anthony Benedetto, Botulinum Toxins in Clinical Aesthetic Practice, Second
Edition, ISBN 9780415476362
Robert Baran and Howard I. Maibach, Textbook of Cosmetic Dermatology,
Fourth Edition, ISBN 9781841847009
Neil Sadick, Diane Berson, Mary P. Lupo, and Zoe Diana Draelos,
Cosmeceutical Science in Clinical Practice, ISBN 9780415471145
Paul Carniol and Gary Monheit, Aesthetic Rejuvenation Challenges and
Solutions: A Global Perspective, ISBN 9780415475600
Avi Shai, Robert Baran, Howard I. Maibach, Handbook of Cosmetic Skin
Care, Second Edition, ISBN 9780415467186
Benjamin Ascher, Marina Landau, and Bernard Rossi, Injection Treatments in
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David J. Goldberg, Laser Hair Removal, Second Edition, ISBN
Paul J. Carniol and Neil S. Sadick, Clinical Procedures in Laser Skin
Rejuvenation, ISBN 9780415414135
C. William Hanke, Gerhard Sattler, and Boris Sommer, Textbook of
Liposuction, ISBN 9781841845326
David J. Goldberg, Fillers in Cosmetic Dermatology, ISBN 9781841845098
Textbook of Cosmetic Dermatology
Fifth Edition

Edited by
Robert Baran, MD
Nail Disease Center
Cannes, France
Howard I. Maibach, MD
Department of Dermatology
University of California San Francisco, School of Medicine
San Francisco, California, U.S.A.
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Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ix

Section I: Skin Science and Parameters

1. Skin Physiology and Gender . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Ethel Tur

2. Climatic Influence on Cosmetic Skin Parameters . . . . . . . . . . . . . . . . . . . . . . . 16

Mathias Rohr and Andreas Schrader

3. Transepidermal Water Loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Jan Kottner and Annika Vogt

4. Nail Penetration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Rania Elkeeb, Xiaoying Hui, and Howard I. Maibach

Section II: Pharmacology of Cosmetic Products and Ingredients

5. Sensitive Skin: New Findings Yield New Insights . . . . . . . . . . . . . . . . . . . . . . 45
Miranda A. Farage and Howard I. Maibach

6. Organic Acids with Novel Functions: Hydroxy, Bionic, N-acetylamino

Acids and N-acylpeptide Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Ruey J. Yu and Eugene J. Van Scott
7. Retinyl Propionate and Related Retinoids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
John E. Oblong

8. Idebenone (Hydroxydecyl Ubiquinone) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Birgit A. Neudecker, Falko Diedrich, and Howard I. Maibach
9. Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Frank Dreher

10. Topical Retinol: An Efficacious Solution for Improvement of

Main Photodamage Signs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Christiane Bertin and Thierry Oddos

11. Applications of Non-Denatured Soy in Skin Care. . . . . . . . . . . . . . . . . . . . . . . 93

Jue-Chen Liu, Jeff Wu, and Miri Seiberg

12. Kinetin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Stanley B. Levy

13. Urokinase and Plasmin in Dry Skin and Skin Aging . . . . . . . . . . . . . . . . . . . 117
Yuji Katsuta

14. Ceramides and the Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

David J. Moore, Clive R. Harding, and Anthony V. Rawlings

15. 4-Hexyl-1,3-Phenylenediol, an NF-kB Inhibitor, Improving

Clinical Signs of Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Cécilia Brun, Simarna Kaur, Michael D Southall, Christiane Bertin,
and Thierry Oddos

16. Perfumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

Jeanne Duus Johansen

17. Alternative and Natural Treatments in Dermatology . . . . . . . . . . . . . . . . . . . 153

Daniel Oxman and Cheryl Levin

Section III: Non-Pathological Skin Treatments

18. Skin Care Products for Normal, Dry, and Greasy Skin . . . . . . . . . . . . . . . . . 167
Christine Lafforgue, Céline Try, Laurence Nicod, and Philippe Humbert

19. Self-Tanning Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

Stanley B. Levy

20. Astringents, Masks, and Ancillary Skin Care Products . . . . . . . . . . . . . . . . . 178

Zoe Diana Draelos

21. Regulatory Overview of Cosmeceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

Lauren A. Hassoun, Howard I. Maibach, and Raja K. Sivamani
22. Photodamage: Protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Laurent Meunier

23. Photodamage and Skin Cancer: How Successful Are Sunscreens as a

Means of Prevention? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Xinyi Du and Douglas Maslin

24. Photodamage: Protection and Reversal with Topical Antioxidants . . . . . . . 199

Karen E. Burke

25. Actinic Keratosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

Brigitte Dréno

26. Safety of UV Nail Lamps as Used in Professional Nail Salons . . . . . . . . . . . 220

Douglas Schoon

Section IV: Specific Locations and Conditions

27. Hair Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
John Gray

28. Dandruff and Seborrheic Dermatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

James R. Schwartz and Thomas L. Dawson, Jr.

29. The Periorbital Wrinkle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

Martin R. Green

30. Cosmetology for Normal Nails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

Robert Baran and Douglas Schoon

31. Cosmetics for Abnormal and Pathological Nails . . . . . . . . . . . . . . . . . . . . . . . 276

Douglas Schoon and Robert Baran

32. Evaluating Hand and Body Lotions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

F. Anthony Simion

33. Anticellulite Products and Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308

Enzo Berardesca

34. Therapy of Telangiectasia and Varicose Veins and Their Complications . . 312
Christian R. Halvorson, Robert A. Weiss, and Margaret A. Weiss

35. Management of Hirsutism and Hypertrichosis . . . . . . . . . . . . . . . . . . . . . . . . 321

Ralph M. Trüeb and Daisy Kopera

36. Pigmentation: Dyschromia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

Thierry Passeron and Jean-Paul Ortonne

37. Treatment of Keloids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349

Joshua E. Lane

38. Keratolytic Treatment of Acne . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

Brigitte Dréno

39. Hidradenitis Suppurativa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369

Emil Knudsen List and Gregor B.E. Jemec

Section V: Specific Groups

40. Age-Related Changes in Male Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
Stefanie Lübberding and Nils Krüger

41. Ethnic Cosmetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384

Enzo Berardesca

42. Ethnic Variation in Hair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390

Nina Otberg

43. Ethnic Differences in Skin Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398

Rishu Gupta and Howard I.Maibach

44. Changes in Female Hair with Aging: New Understanding and Measures . . 413
Paradi Mirmirani, R. Scott Youngquist, and Thomas L. Dawson, Jr.

45. Menopause, Skin, and Cosmetology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424

Michel Faure and Evelyne Drapier-Faure

Section VI: Cosmetological Treatments

46. Mesotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
Maria Pia De Padova, Gabriella Fabbrocini, Sara Cacciapuoti,
and Antonella Tosti

47. Microneedles and Cosmetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436

Raja K. Sivamani and Howard I. Maibach

48. Photodynamic Therapy in Dermatology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .442

Jacques Savary

49. Cosmetic Cryotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450

Eshini Perera, Poorna Weerasinghe, and Rodney Sinclair

50. Botulinum Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

Doris Hexsel

51. Soft Tissue Augmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473

Kathleen Sikora Viscusi and C. William Hanke

52. Bioelectricity and Its Application in Cosmetic Dermatology . . . . . . . . . . . . 481

Ying Sun and Jue-Chen Liu

53. Chemical Peels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498

Philippe Deprez

54. Lasers and Light Sources for Vascular and Pigmented Components
of Photoaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
Anne Marie Mahoney and Robert A. Weiss

55. Nonablative Laser Rejuvenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519

Christian R. Halvorson, Karen L. Beasley, and Robert A. Weiss

56. Cryolipolysis for Non-Surgical Fat Reduction . . . . . . . . . . . . . . . . . . . . . . . . . 535

Christine C. Dierickx

Section VII: Assessment Techniques

57. Using the Behind-the-Knee Test to Evaluate Lotion Transfer
from Products to Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
Miranda A. Farage

58. Assessing the Efficacy of Moisturizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561

Whitney Hannon

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585

Robert Baran Nail Disease Center, Cannes, France

Karen L. Beasley Department of Dermatology, University of Maryland School of

Medicine, Baltimore, Maryland; and Maryland Laser, Skin & Vein Institute, Hunt
Valley, Maryland

Enzo Berardesca San Gallicano Dermatological Institute, Rome, Italy

Christiane Bertin Johnson & Johnson Group of Consumer Companies, Skin Care
Research Institute, Issy les Moulineaux, France

Cécilia Brun Johnson & Johnson Skin Research Center, Johnson & Johnson Santé
Beauté France, Val de Reuil, France

Karen E. Burke Department of Dermatology, The Mount Sinai Medical Center,

New York, New York

Sara Cacciapuoti Department of Dermatology, University of Naples, Napoli, Italy

Thomas L. Dawson, Jr. Agency for Science, Technology, and Research (A*STAR),
Institute of Medical Biology, Singapore

Philippe Deprez Clinica HERA, Empuriabrava, Spain

Falko Diedrich Private practice, München, Germany

Christine C. Dierickx Skinperium Clinic, Boom, Belgium

Zoe Diana Draelos Department of Dermatology, Duke University School of

Medicine, Durham, North Carolina

Frank Dreher NEOCUTIS, a Division of MERZ North America, Inc., San Mateo,

Brigitte Dréno Department of Dermatology, University Hospital Hotel Dieu,

Nantes, France

Xinyi Du University of Cambridge, Cambridge, United Kingdom

Rania Elkeeb Department of Dermatology, University of California,

San Francisco, San Francisco, California

Gabriella Fabbrocini Department of Dermatology, University of Naples, Napoli,


Miranda A. Farage The Procter and Gamble Company, Cincinnati, Ohio

Evelyne Drapier-Faure Edouard Herriot Hospital, Lyon, France

Michel Faure Department of Dermatology, University of Orléans, Orléans, France

John Gray Procter & Gamble Technical Centres Limited, Egham, United Kingdom

Martin R. Green Unilever Research, Colworth Science Park, Sharnbrook,

United  Kingdom

Rishu Gupta Keck School of Medicine, University of Southern California, Los

Angeles, California; and Department of Dermatology University of California, San
Francisco, San Francisco, California

Christian R. Halvorson MD Laser, Skin & Vein Institute, Hunt Valley, Maryland

C. William Hanke Laser and Skin Surgery Center of Indiana, St. Vincent’s
Hospital, Carmel, Indiana

Whitney Hannon Private practice, Seattle, Washington

Clive R. Harding Unilever Research Port Sunlight Laboratory, Wirral, United


Lauren A. Hassoun School of Medicine, University of California—Davis,

Sacramento, California

Doris Hexsel Department of Dermatology, Pontificia Universidade Catolica do

Rio Grande do Sul, Porto Alegre, Brazil

Xiaoying Hui Department of Dermatology, University of California San

Francisco, San Francisco, California

Philippe Humbert Department of Dermatology, University Hospital Saint-

Jacques, Besançon, France

Gregor B.E. Jemec Department of Dermatology, Roskilde Hospital, Health

Sciences Faculty, University of Copenhagen, Copenhagen, Denmark

Jeanne Duus Johansen National Allergy Research Centre, Department of

Dermato-Allergology, Gentofte Hospital, University of Copenhagen, Hellerup,

Yuji Katsuta Shiseido Global Innovation Center, Yokohama, Japan

Simarna Kaur Johnson & Johnson Skin Research Center, CPPW, A Division of
Johnson & Johnson Consumer Companies, Inc., Skillman, New Jersey

Daisy Kopera Center of Aesthetic Medicine, Department of Dermatology,

Medical University Graz, Graz, Austria

Jan Kottner Clinical Research Center for Hair and Skin Science, Department of
Dermatology and Allergy, Charité-Universitätsmedizin Berlin, Berlin, Germany

Nils Krüger Rosenpark Research, Darmstadt, Germany

Christine Lafforgue Dermo–Pharmaco & Cosmeto, Châtenay-Malabry, France

Joshua E. Lane Department of Surgery, Division of Dermatology, Department of

Internal Medicine, Mercer University School of Medicine, Macon, Georgia; and
Division of Dermatology Department of Medicine, The Medical College of Georgia,
Augusta, Georgia; and Department of Dermatology, Emory University School of
Medicine, Atlanta, Georgia

Cheryl Levin Harvard Vanguard Medical Associates, Department of

Dermatology, Boston, Massachusetts

Stanley B. Levy Duke University School of Medicine, Durham, North Carolina


Jue-Chen Liu Liu Consulting LLC, New York, New York

Emil Knudsen List Department of Dermatology, Roskilde Hospital, Health

Sciences Faculty, University of Copenhagen, Copenhagen, Denmark

Stefanie Lübberding Rosenpark Research, Darmstadt, Germany

Anne Marie Mahoney Maryland Laser, Skin and Vein Institute, Hunt Valley,

Howard I. Maibach Department of Dermatology, University of California San

Francisco, San Francisco, California

Douglas Maslin Addenbrooke’s Hospital, Cambridge, United Kingdom

Laurent Meunier Department of Dermatology, University of Montpellier, Nimes,


Paradi Mirmirani Department of Dermatology, The Permanente Medical Group,

Vallejo, California

David J. Moore GSK, Research Triangle Park, North Carolina

Birgit A. Neudecker Department of Dermatology, University of California

San Francisco, School of Medicine, San Francisco, California

Laurence Nicod Cellular Biology and Genetic Laboratory, University Hospital

Saint-Jacques, Besançon, France

John E. Oblong The Procter & Gamble Company, Miami Valley Laboratories,
Cincinnati, Ohio

Thierry Oddos Johnson & Johnson Skin Research Center, Johnson & Johnson
Santé Beauté France, Val de Reuil, France

Jean-Paul Ortonne Department of Dermatology, University Hospital of Nice,


Nina Otberg Skin and Laser Center Potsdam, Hair Clinic, Potsdam and Hair
Transplant Center Berlin–Potsdam, Berlin, Germany

Daniel Oxman University of Minnesota, School of Medicine, Duluth, Minnesota

Maria Pia De Padova Department of Dermatology, Nigrisoli Hospital, Bologna,


Thierry Passeron Department of Dermatology, University Hospital of Nice, Nice,


Eshini Perera The University of Melbourne, Melbourne, Australia

Anthony V. Rawlings AVR Consulting Ltd, Northwich, United Kingdom

Mathias Rohr Institut Dr. Schrader Hautphysiologie, Holzminden, Germany

Jacques Savary Private practice, Paris, France

Douglas Schoon Science & Technology, Creative Nail Design, Inc., Vista,

Andreas Schrader Institut Dr. Schrader Hautphysiologie, Holzminden, Germany


James R. Schwartz The Procter & Gamble Company, Beauty Care Product
Development, Cincinnati, Ohio

Miri Seiberg Seiberg Consulting, LLC, Princeton, New Jersey

F. Anthony Simion Kao USA, Cincinnati, Ohio

Rodney Sinclair Department of Dermatology, University of Melbourne and

St. Vincent’s Hospital, Melbourne, Australia

Raja K. Sivamani Department of Dermatology, University of California—Davis,

Sacramento, California

Michael D. Southall Johnson & Johnson Skin Research Center, CPPW, A Division
of Johnson & Johnson Consumer Companies, Inc., Skillman, New Jersey

Ying Sun Johnson & Johnson Consumer Personal Group, Skillman, New Jersey

Antonella Tosti Department of Dermatology and Cutaneous Surgery, University

of Miami, Miami, Florida

Ralph M. Trüeb Center for Dermatology and Hair Diseases, Zurich, Switzerland

Céline Try Department of Dermatology, University Hospital Saint-Jacques,

Besançon, France

Ethel Tur Department of Dermatology, Sackler School of Medicine, Tel Aviv

University, Tel Aviv, Israel

Eugene J. Van Scott Private practice, Abington, Pennsylvania

Kathleen Sikora Viscusi Dermatology Consultants, Marietta, Georgia

Annika Vogt Clinical Research Center for Hair and Skin Science, Department of
Dermatology and Allergy, Charité-Universitätsmedizin Berlin, Berlin, Germany

Margaret A. Weiss Department of Dermatology, University of Maryland School

of Medicine, Baltimore, Maryland; and Laser Skin & Vein Institute Hunt Valley,

Poorna Weerasinghe Department of Dermatology, University of Melbourne,

Melbourne, Australia

Robert A. Weiss Department of Dermatology, University of Maryland School of

Medicine, Baltimore, Marylandand; and Laser Skin & Vein Institute Hunt Valley,

Jeff Wu Johnson & Johnson Consumer Personal Group, Skillman, New Jersey

R. Scott Youngquist The Procter & Gamble Company, Mason Business Center,
Mason, Ohio

Ruey J. Yu Private practice, Chalfont, Pennsylvania

Section I
Skin Science and Parameters

Skin Physiology and Gender

Ethel Tur

INTRODUCTION Forearm skinfold thickness, as measured by a caliper,

Many characteristics of the body are reflected in the skin, decreases starting at age 35 for women and 45 for men. Starting
gender being a prominent one. Genetic and hormonal differ- at age 35, it is thinner in women than in men (12). In younger
ences affect skin structure and function, resulting in variations subjects 17–24 years, forearm, thigh, and calf skinfold thick-
between women and men and causing these gender varia- ness in women is lower than in men (13).
tions to change with age. In addition, exogenous factors differ Heel pad thickness, an indicator of soft tissue thickness
according to differences in lifestyle between the sexes. in the body, was thicker in Ethiopian men than in women (14).
During the last few decades, methodologies used in der- Skinfold compressibility in Japanese students was greater in
matological research have improved substantially, providing women than in men at the pectoral site, and smaller at nuchal,
means of objective evaluation of skin function and characteris- submental, biceps, thigh, suprapatellar, and medial calf sites
tics. The number of studies addressing various aspects of dif- (7). The changes in the distribution of fat between the ages of 6
ferences between women and men has increased in the last few to 18 years were studied in 2300 subjects (15). Up to 12 years of
years along with the growing interest in studying gender-related age, there was no difference between the two sexes: the mass
differences of physiological and disease processes (1,2). However, of the subcutaneous fat increased more than threefold, while
the subject has not yet been systematically studied, so much of that of the internal mass increased less than twice. After the
the data are by-products of studies with a different focus. This age of 12, the relative mass of the subcutaneous fat continued
chapter outlines the various aspects of physiological differences to increase in girls but not in boys.
between the skin of women and men, based on the available data. The distribution of fat over the body is different in men
and women (16). In men, an increase in fat tends to accumu-
late in the abdominal region and upper parts of the body,
STRUCTURAL AND ANATOMICAL whereas in women it is located in the lower body, particularly
CHARACTERISTICS (TABLE 1.1) in the gluteal and femoral regions. In addition, the proportion
The skin of female frogs is thicker than that of males in all body of body fat is higher in non-obese women than in non-obese
regions (3) (the opposite is true for rat skin[4]). In humans, skin men. The characteristic difference in body fat distribution
thickness (epidermis and dermis) is greater in men than in between the sexes exists both in non-obese and obese subjects.
women (5), up to 1.428 times (6), whereas the subcutaneous fat Lipoprotein lipase activity and mRNA levels were higher in
thickness is greater in women (7). The skin of men is thicker women in both the gluteal and abdominal regions. In women,
across the entire age range of 5–90 years (8). Hormonal influence higher enzyme activity was found in the gluteus than in the
on skin thickness was demonstrated when conjugated estrogens abdomen, whereas in men it was higher in the abdomen.
were given to postmenopausal women (9). Following 12 months’ These regional and sex differences in lipoprotein lipase activ-
therapy, the dermis was significantly thicker, and histologic ity might underlie the difference in fat distribution and total
improvement in the previously atrophic epidermis was noted. fat content. Variation is at both the mRNA level and post-
Epidermal thickness alone, as measured by optical coherence translational level.
tomography, does not differ between men and women, except
for the forehead epidermis which is thinner in women (10).
Skin collagen and collagen density were measured in BIOCHEMICAL COMPOSITION (TABLE 1.2)
addition to dermal thickness (11). The skin of men demon- Significant age-related differences in the stratum corneum
strated a gradual thinning with advancing age (12–93 years), sphingolipid composition were found in women, but not
whereas the thickness of women’s skin remained constant up in men (17). From prepubertal age to adulthood there was
until the fifth decade, after which it decreased with age. The a  significant increase in ceramide 1 and 2 accompanied by
male forearm skin contained more collagen at all ages in the a decrease in ceramide 3 and 6. After maturity there was a
range 15–93 years. In both sexes there was a linear decrease in decrease in ceramide 2 and an increase in ceramide 3. These
skin collagen with age. Collagen density calculated as the ratio findings indicate an influence of female hormones on the com-
of skin collagen to thickness was lower in women at all ages. position of stratum corneum sphingolipids. These lipids play
The rate of collagen loss was similar in both sexes. Women an important role in the water permeability barrier function of
start with lower collagen content; therefore they seem to age the human epidermis, and thus endocrinological factors may
earlier than men. Collagen density, representing the packing of influence this barrier.
fibrils in the dermis, is lower in women than in men. This may Human tissue kallikreins are a family of 15 trypsin or
be due to androgen, since skin collagen density is increased in chymotrypsin-like secreted serine proteases (hK1-hK15). hK5,
patients with virilism. hK6, hK7, hK8, and hK13 have been identified in the stratum

Table 1.1 Structural and anatomical characteristics

Ref. Finding Obtained by Subjects Conclusions
(a) Significant differences
10 Forehead epidermis thinner in women Optical coherence 83 Caucasians;
Other sites: Epidermal thickness does not differ tomography Young: 20–40 y
between men and women Old: 60–80 y
5 Skin thickness in humans greater in men than in Echographic 24 women; 24 men;
women, except for lower back in young evaluation half 27–31 y
subjects half 60–90 y
8 Men’s skin thicker than women’s across the Ultrasonic 69 women; 54 men;
entire age range of 5–90 y echography; 5–90 y
6 Men’s skin thicker than women’s, up to 1.438 12.0-MHz- in 112 healthy;
times B-mode 43 women; 69 men;
19–28 years;
24 sites
9 Thickening of dermis following 12 months Conjugated 28 estrogen; Estrogens affect skin
estrogen therapy estrogen therapy; 26 placebo; thickness
ultrasound women: 51–71 y
11 Men: Gradual thinning of skin with advancing Skin collagen, skin Collagen: Rate of collagen loss
age thickness and 80 women; 79 men; same in men and
Women: Thickness constant up to 5th decade, collagen density, 15–93 y women, although total
then decreasing with age measured Thickness: skin collagen content is
chemically and 107 women; 90 men; less in women than
histologically 12–93 y men at all ages
26 women; 27 men;
15–93 y
12 Forearm skinfold thickness decreases starting at Caliper; forearm 145 women and men;
age 35 for women and 45 for men 8–89 y
Starting at age 35 it is thinner in women than in
13 Skinfold thickness lower in women Caliper; forearm, 42 women; 37 men;
thigh, and calf 17–24 y
7 Subcutaneous fat thickness greater in women Caliper and 45 women; 41 men;
ultrasound Japanese; 18–22 y
14 Heel pad thickness thicker in men than in Ankle x-ray 113 women; 125 men;
women; correlation with body weight Ethiopian; 10–70 y
7 Skinfold compression in women is greater in the Caliper and 45 women; 41 men;
trunk and lower in the limbs ultrasound Japanese; 18–22 y
15 Up to 12 years of age no difference between the Caliper 1292 women; 1008 men;
sexes ages 6, 8, 10, 18
Subcutaneous fat increases more than threefold,
while internal fat mass increases less than
After 12 y, the relative mass of the subcutaneous
fat increased in girls but not in boys
16 Lipoprotein lipase activity higher in women Lipoprotein lipase 8 women; 11 men; Regional and sex
Women: Higher values in gluteus than abdomen activity and 37 ± 4 y differences in lipoprotein
Men: Higher in abdomen mRNA levels lipase activity might
measured; underlie the difference
hybridization, in fat distribution and
Northern blot total fat content
Variation is both at mRNA
and post-translational
(b) No significant differences
15 Up to 12 y: The mass of the subcutaneous fat Caliper 1292 women; 1008 men;
increases more than threefold, while that of ages 6, 8, 10, 18
the internal mass increases less than twice in
both sexes

Table 1.2 Biochemical composition

Ref. Finding Obtained by Subjects Conclusions
Significant differences
17 Stratum corneum sphingolipid composition Ethanolic extracts; 27 women; 26 men; Female hormones
differs with age in women but not in men biochemical methods 10–79 y influence the
of lipid identification composition of stratum
corneum sphingolipids
19 Women: Higher concentrations of metals in Liquid chromatography; 60 women; 72 men;
hair trace metal 6–40 y
Concentrations of copper did not differ with determination
age in men, whereas in women they
increased with age

corneum (SC), stratum granulosum, and skin appendages. measuring the speed of dermal–epidermal separation utiliz-
HK6 and hK14 were significantly lower in women between 20 ing the time required for blisters to form by controlled suc-
and 59 y (18). tion (30). From 15 up to 69 years of age, women exhibited
Differences in the metal content of human hair were longer blistering times than men in both antecubital and
found between men and women: higher concentrations of abdominal sites. The difference was more pronounced in the
metals were noted in women. Concentrations of copper did not age range 15–39 years than 40–69 years, and disappeared in
differ with age in men, whereas an increase with increased age older ages.
was noted in women (19). Skin elasticity did not differ between the sexes, as mea-
sured utilizing two suction cup methods (24,31). Similarly, tor-
sional extensibility of the skin, as measured by a twistomenter,
MECHANICAL PROPERTIES (TABLE 1.3) did not differ between the sexes (8).
Clinical assessment, as well as objective measurements of stra- Cutaneous extensibility was identical in men and
tum corneum hydration, and grading of scaling (by adhesive women, but after hydration it increased only in women (32).
tape strippings followed by densitometry readings) showed no Hydration changes the properties of the stratum corneum,
differences between men and women (20). A positive effect of softening it, thus allowing the difference in dermal thick-
estrogens on stratum corneum hydration and wrinkles was ness to express itself as a difference in extensibility. Since
demonstrated when estriol or estradiol cream was applied on the dermis is thinner in women, elimination of the stratum
the face of perimenopausal women (21). corneum factor allows a rapid extensibility of the skin in
The degree of facial wrinkling is affected by gender. In women.
men, forehead wrinkles were increased in all age groups as Plasticity was found to be greater in women than in men
compared with women. However, no gender-dependent dif- in three sites of the foot in one study (33).
ferences were found in upper eyelid wrinkles. Other facial
wrinkles were greater in men than in women in all except the
oldest group (65–75 years), in which wrinkles in women were FUNCTIONAL DIFFERENCES (TABLE 1.4)
greater than or equal to those in men (22). Following pilocarpine iontophoresis, sweat secretion rates
Photographs and dermal elasticity measurement by were higher in men than in women in both healthy and chronic
cutometer showed that the morphology, areas of sagging, and renal failure subjects (26).
elasticity in male faces are similar to those in females in the Body sweat distribution over the upper body in nine
cheek, but sagging at the lower eyelid is more severe in males clothed male and female runners of equal fitness while run-
after middle age (23). ning at 65% and subsequent 15-min rest in a moderate climate
Epidermal hydration affects the friction between the skin (25° C, 53% rh) was investigated using technical absorbent
and textiles. Friction of women showed higher moisture sensi- materials to collect the sweat produced. Local sweat rates
tivity than men, when measured at different hydration states, were higher in men for the mid-front, sides, and mid lateral
when forearm skin was rubbed with dry to completely wet back as compared to women. Both sexes showed similar
textile. Higher skin hydration caused gender-specific changes sweat distribution patterns over the upper body with some
in its mechanical properties and surface properties, leading to exceptions. Men showed higher relative (local to overall)
softening and increased contact area (24). sweat rates than women for the mid lateral back, while it was
Other studies showed no difference of frictional proper- lower for the upper arm, lateral lower back, and upper cen-
ties of the skin, as well as stratum corneum hydration, between tral back. Sweating in both sexes was highest along the spine,
men and women, in both young and old subjects (25,26,27). and higher on the back as a whole than the chest as a whole.
In addition, transepidermal water loss showed no difference Upper arm sweat rate was lowest. Men showed a higher ratio
between the two sexes. In contrast, another study (28) found of highest to lowest local sweat rates (34).
lower basal transepidermal water loss values in women com- Increases in sweating as a function of increasing con-
pared with men aged 18–39 years. centration of acetylcholine significantly differed between
The adhesion of the stratum corneum, measured in males and females. Maximum values were lower in females in
vitro in skin biopsy samples, did not differ between men and response to acetylcholine (35).
women in several body regions (29). But age (and probably The fatty acid composition of sebum is affected by
hormonal) related differences were demonstrated in vivo by androgens in both sexes (36).

Table 1.3 Mechanical properties

Ref. Finding Obtained by Subjects Conclusions
(a) Significant differences
30 From 15 y to 69 y women exhibited Measuring the speed of 178 women, 15–101 y
longer blistering times than men dermal–epidermal 209 men, 16–96 y
The difference was more separation utilizing the time
pronounced in the age range required for blisters to form
15–39 y than 40–69 y, and by controlled suction;
disappeared in older ages antecubital and abdominal
24 Friction of women showed higher Corneometry 11 women Higher skin hydration
moisture sensitivity than men Forearm skin 11 men causes gender
Rubbing with various hydration specific changes in
states, dry to wet textile its mechanical
properties, leading to
softening and
increased contact
22 Men: Increased forehead wrinkles Photographs: Replicas from 173 Japanese men and Men tend to have more
compared with women; no five facial sites used to women severe wrinkles than
differences in upper eyelid measure surface roughness women
wrinkles This tendency
Other facial wrinkles were greater in disappeared or was
men than in women in all except reversed in some
the oldest group (age, 65–75 y), in regions of the face
which wrinkles in women were and in individuals
greater than or equal to those in more than 60 y old.
23 Sagging in male faces: Similar to Photograph-based grading, 98 Japanese men, 108 Dermal elasticity of
females in the cheek, but sagging cutometer women male facial skin
at the lower eyelid is more severe 20–60 y decreased with age
in males after middle age similar to that of
females, except for
the lower eyelids
(b) No significant differences
20 Stratum corneum hydration, and Clinical assessment and 50 women; 22 men;
grading of scaling showed no bioengineering 21–61 y
differences between men and measurement
21 A positive effect of estrogens on Stratum corneum hydration 18 women (8 applied Topical treatment with
facial skin: Moisture increased, and wrinkles– profilometry estriol, 10 estradiol) estrogen seems
wrinkles decreased of skin replicas 46–66 y promising
25 No difference between men and Bioengineering measurement 7 women, 25 y (mean)
women in friction, moisture, 7 men, 29 y; 7 women,
transepidermal water loss 75 y; 8 men, 74 y
26 No difference in moisture Bioengineering; healthy and Healthy: 24 women, 21
chronic renal failure subjects men
Patients: 30 women, 50
31 Skin elasticity did not differ between In vivo suction device Young: 8 women (26 y);
the sexes, as measured by suction (bioengineering) 8 men (28 y)
devices Old: 9 women (75 y);
8 men (75 y)
24 Skin viscoelasticity comparable for Suction chamber; forearm 11 women, 11 men
women and men skin; rubbing with various
hydration states, dry to wet
8 Torsional extensibility did not differ Twistometer 69 women; 54
between men and women men
5–90 y
29 The adhesion of the stratum Biopsy; in vitro measurement 9–34 women and men
corneum did not differ between of the force needed to (number varied with
men and women separate cells site studied)
20–40 y

Table 1.4 Functional differences

Ref. Finding Obtained by Subjects Conclusions
Significant differences
26 Men sweat more than women Pilocarpine Healthy: 24 women;
iontophoresis – 21 men
healthy and chronic CRF patients: 30
renal failure subjects women; 50 men;
18–75 y
34 Local sweat rates higher in men for the Technical absorbent 9 clothed male and
mid-front, sides, and mid lateral back materials to collect the female runners while
Men showed higher relative (local to overall) sweat produced in a running at 65% and
sweat rates than women for the mid moderate climate (25 subsequent 15-min
lateral back, while it was lower for the degrees C, 53% rh) rest
upper arm, lateral lower back, and upper
central back
32 Cutaneous extensibility increased only in Bioengineering 15 women; 14 men Hydration allows the effect
women after hydration methods 23–49 y and 60–93 y of thinner dermis in
women to be reflected
in extensibility
35 Increases in sweating with increasing Intradermal 12 women, 12 men Peripheral modulation of
concentration of acetylcholine significantly microdialysis sudomotor activity in
differed between men and women females
Maximum values were lower in women in
response to acetylcholine

Sex-related differences in the metabolism in the skin CUTANEOUS MICROVASCULATURE

of  topically applied compounds were found in guinea pig (TABLE 1.6)
skin (37). Hormonal factors affect the skin blood flow: differences
between men and women were found during the reproduc-
tive years, and differences were found within different phases
DIFFERENCES IN RESPONSE of the menstrual cycle (42). Moreover, vasospastic diseases,
TO IRRITANTS (TABLE 1.5) such as Raynaud’s phenomenon, are more common in women,
The incidence of irritant dermatitis is higher in women than more prevalent in the reproductive years, and improve during
in men, but experimental irritant dermatitis does not differ pregnancy, suggesting an influence of female sex hormones
between men and women (38,39). Occupational factors lead- (43). Skin circulation varied during the menstrual cycle. There
ing to a greater exposure to irritants by women may pro- might be a direct influence of sex hormones on the blood vessel
vide an explanation of this discrepancy. In a study of skin wall or an indirect systemic hormonal action causing a cyclic
irritability by sodium lauryl sulfate, women showed lower pattern in women. Estrogens influence the sympathetic ner-
baseline transepidermal water loss compared with men, vous system, inducing an upregulation of (vasoconstrictive)
but after irritation both sexes gave similar transepidermal α2-adrenoceptors. Thus blood flow measurements utilizing
water loss values (28). The importance of interpretation of laser Doppler flowmetry revealed a reduction of basal cutane-
the results, and the lack of a standardized way of analyzing ous blood flow in women compared with men (43,44,45), but
them, is illustrated in the latter study. The authors define an these differences existed only in young women and not in
irritation index as the ratio of the difference between the val- women over 50 years (46). This reduction was due to a basal
ues for irritated and non irritated skin to the value for non increase in sympathetic tone rather than to a local structural or
irritated skin. Although the value for irritated skin did not functional difference in the cutaneous circulation.
differ between men and women, this index was higher in The vasodilatation induced by local heating occurred at
women, since the value for non irritated skin was lower in a lower skin temperature in women (47). However, the maxi-
men, and so the authors conclude that women’s skin is more mum skin blood flow following heating of the skin was not
irritable. A review article considering the absolute values different between men and women, and neither was the post-
following irritation interpreted the same results as indicat- occlusive reactive hyperemia response in a study including a
ing no sex-related differences in sodium lauryl sulfate irri- group of women aged 20–59 years (43). In contrast, in a study
tation.38 Until a universal way of interpreting the results is that divided women according to age, the reactive hyperemia
established, contradictory conclusions may be reached by response was lower in young women compared both with
different analyses of the same set of data. In another study, women over 50 years and with young men (46). The latter
baseline transepidermal water loss did not differ between study also measured the response to cooling, which was pro-
men and women (40). This study found no significant dif- longed in young women compared with the other two groups.
ferences between men and women in developing cumula- Skin microvascular response to vasodilators was evalu-
tive irritant dermatitis when visual scoring, transepidermal ated by laser Doppler perfusion imager, an instrument that
water loss, skin blood flow, and dielectric water content were maps the skin blood perfusion. The substances used were ace-
assessed. Changes during the menstrual cycle, however, tylcholine, an endothelium-dependent vasodilator, and nitro-
were demonstrated by measuring baseline transepidermal prusside and isoprenaline–two endothelium-independent
water loss (41). vasodilators with different modes of action. The substances

Table 1.5 Irritants

Ref. Finding Obtained by Subjects Conclusions
(a) Significant differences
38 Incidence of irritant dermatitis higher in Occupational factors
women than in men
28 Lower baseline transepidermal water loss Sodium lauryl sulfate 15 women; 23 men; Comparing the irritation
in women compared with men, but after irritation; evaporimeter 18–39 y index (the difference
irritation similar values in both sexes between irritated and
unirritated values over
unirritated): female skin
more irritable
41 Higher on the day of minimal estrogen/ Back and forearm sites; 9 women; Barrier function is less
progesterone secretion compared with baseline 19–46 y (mean 32) complete just prior to
the day of maximal secretion transepidermal water the onset of menses
Also higher on the day of maximal loss; evaporimeter compared with the days
progesterone secretion compared with just prior to ovulation
the day of maximal estrogen secretion
(b) No significant differences
39 No significant differences between men Irritation tested for 11 21 women; 21 men with No tendency to stronger
and women with or without hand irritants at several hand eczema; reactions in either sex
eczema concentrations 21 women; 21 Speculation:
men without hand Women’s occupations
eczema; lead to a greater
20–60 y exposure to irritants
40 No significant differences between men Repeated once-daily 7 women; 7 men; No sex-related
and women in developing cumulative application of 3 16–65 y susceptibility to
irritant dermatitis concentrations of develop cumulative
irritant (SLS), 5 days, irritant dermatitis.
followed by a patch Speculation:
test; upper back; Women’s occupational
bioengineering and domestic duties
measurements lead to a greater
exposure to irritants

were iontophorized into the skin. The response to nitroprus- with removal of a tonic vasoconstrictor effect of endothelin-B.
side, and to a lesser extent to acetylcholine, was higher in In women, it caused a vasoconstriction, demonstrating release
women before menopause than after (48), reflecting functional of tonic vasodilator activity (53).
and structural changes in skin vasculature with aging.
The cutaneous blood flow response to topical and intra-
dermal administration of histamine was comparable in men SENSORY FUNCTIONS (TABLE 1.7)
and women at three anatomical sites: the back, the volar side of Thermoregulatory Response
the forearm, and the ankle (49). These observations indicate that Studies of human thermoregulation were conducted by expos-
there are no functional differences between men and women in ing subjects to various thermal environments. The importance
the skin microvascular response to histamine. However, hista- of taking into account all the possible variables is demon-
mine administered by iontophoresis produced bigger wheals strated in studies of the physiological responses to heat stress
in women, as measured by laser Doppler flowmetry (44). The (54): data showed differences between women and men. But
bigger wheals were attributed to differences in the stratum cor- when taking into consideration the differences in the percent-
neum layer, which is the main obstacle to penetration. age of fat in the body and the ratio between the body surface
Transcutaneous oxygen pressure is a method that measures and mass, the effect of gender disappeared.
changes in oxygen pressure at the skin surface that are mainly In contrast to these results of heat stress, the response of
determined by changes in skin blood flow. During skin surface Japanese young subjects to cold stress differed with gender,
measurement, significantly higher values of transcutaneous although body surface area-to-mass ratios were similar (55).
oxygen pressure were noted in women (50,51). The difference Subjects were exposed to cold (12°C) for 1 hour at rest in sum-
might be explained by the thinner epidermis of women. Age- mer and in winter. In winter, women’s tolerance to cold was
related sex differences were noted in measuring transcutane- superior to men’s, whereas no significant differences between
ous oxygen pressure during postocclusive reactive hyperemia. the sexes were found in the summer. The differences in cold
Greater values were found in adult women than in men, but no tolerance may be caused by differences in the distribution of
differences were found between boys and girls (52). fat over the body, even though body surface area-to-mass ratios
The contribution of endothelin-B receptors to resting were similar in the two sexes.
cutaneous vascular tone differs between men and women. The thermal sensitivity distribution (topographical
In men, endothelin-B receptors mediate vasoconstriction, mapping) over the glabrous skin of the hand in men and in
whereas in women, endothelin-B receptors mediate vasodi- women was assessed by measuring warm and cold thresholds
lation. Blockade of endothelin-B receptors by a competitive in 25 healthy volunteers (12 women, 13 men), applying a multi-
antagonist (BQ-788) in men caused skin vasodilation consistent site test of 23 locations on the volar part of the hand. The palm

Table 1.6 Cutaneous microcirculation

Ref. Finding Obtained by Subjects Conclusions
(a1) Significant differences
43 Reduction in basal skin blood flow in Bioengineering 56 women; 44 men;
women measurement 20–59 y
45 Reduction in facial basal skin blood flow Laser Doppler 5 women; 5 men;
in women 25–52 y
44 Reduction in basal skin blood flow in Bioengineering 26 women; 23 men; Sympathetic tone is
women measurement; cooling 23–38 y Increased, not a
and warming to structural or functional
change sympathetic difference in the
tone cutaneous circulation
42 Skin circulation varied during menstrual Bioengineering 31 women; 15–45 y Skin blood flow and its
cycle: Basal flow lowest in the luteal measurements at 4 response to cold varies
phase, highest in the pre-ovulatory times during the during the menstrual
phase menstrual cycle cycle
Greatest cold-induced constriction and
lowest recovery in the luteal phase
46 Reactive hyperemia response lower in Bioengineering 12 women, 19–39 y Hormonal factors might
young women as compared to both measurement; 13 women, 51–67 y explain the differences
women over 50 y or young men postocclusive reactive 13 men, 22–47 y Different dressing habits
Response to cooling prolonged in young hyperemia and direct may also contribute
women compared with the other two and indirect cooling
47 Vasodilatation induced by local heating Bioengineering 9 women; 6 men;
occurs at a lower skin temperature in measurement age not specified
48 Response to nitroprusside higher in Laser Doppler perfusion 21 women; 13 men; Indicating functional and
women before menopause than after imager; iontophoresis 18–80 y structural changes in
skin vasculature of
women with aging
4 Histamine produced bigger wheals in Histamine administered 33 women; 38 men; Differences in the stratum
women by iontophoresis 15–52 y corneum layer
53 Endothelin-B receptors mediate Laser Doppler, 11 women; 11 men; Resting tone is different in
vasoconstriction in men and microdialysis 33± 3 women; women and men
vasodilatation in women 30± 3 men
(a2) Significant differences: Transcutaneous oxygen pressure
50 Significantly higher values of Bioengineering; anterior 18 women; 42 men;
transcutaneous oxygen pressure in chest, forearm 22–88 y
51 Significantly higher values of Bioengineering; 23 sites 7 women; 12 men; Might be explained by
transcutaneous oxygen pressure in on face, extremities, 21–63 y women’s thinner
women and trunk epidermis
52 Transcutaneous oxygen pressure during Bioengineering Adults: Hormonal influence is
postocclusive reactive hyperemia measurement; 30 women; 37 indicated
greater in adult women than in men, but forearm; men;
did not differ between boys and girls postocclusive reactive 22–60 y
hyperemia, 35–37°C Children before
puberty: 34
(b) No significant differences
49 No difference in cutaneous blood flow Topical and intradermal 10 women; 10 men;
response to histamine administration; 24–34 y
43 No difference in postocclusive reactive Bioengineering 56 women; 44 men;
hyperemia and maximum skin blood methods 20–59 y
flow following heating

area was more sensitive than the fingers to both warm and cold women were tested twice, once in the follicular and once in the
stimuli. On the palm itself, the proximal part was the most sen- luteal phase of the menstrual cycle. Blood flow was measured
sitive. Women were more sensitive than men to both warm and before and during local cooling of one hand at 15° C. Local cool-
cold sensations (56). ing evoked a significantly greater decrease in cutaneous blood
Cold-induced vasomotor response was measured by laser flow in women than in men in direct as well as in indirect
Doppler flowmetry in 12 healthy men and 12 healthy women. response conditions. Direct response to local cooling was signif-
Both direct response (at the site of cooling) and indirect response icantly greater in the luteal phase than in the follicular phase. In
(at a site remote from the cooling site) were measured (57). The contrast, there was no menstrual-cycle–dependent difference in

Table 1.7 Sensory function

Ref. Finding Obtained by Subjects Conclusions
(a) Significant differences
61 Women more sensitive to small Marstock 67 women; 83 men;
temperature changes and to method–quantitative 10–73 y
pain caused by either heat or
62 Lower threshold values in Pricking pain sensation 93 women; 165 men;
women than in men to heat; threshold 18–28 y
determination, volar 132 women; 135 men;
forearm 50–90 y
63 Women more sensitive than Pressure threshold 68 women; 68 men;
men: Palm and sole, but not measurement; palm, 17–30 y
on the forearm sole, forearm
64 Neonate girls: Significantly Skin conductance 20 women; 20 men; These differences may represent
higher conductance than (autonomic function) neonates: 60–110 h differences in maturation
boys Very young: No effect yet of training
and different behavior accorded
the sexes
55 Women’s tolerance to cold Exposed to cold (12°C) 7 women; 8 men; Differences in fat distribution over
superior to men’s in winter for 1 h at rest in Japanese; 18–26 y the body, even though body
summer and in surface area-to-mass ratios were
winter; skin and similar in the two sexes, might
body temperature have contributed to the
differences in cold tolerance
59 Greater decrease in women in Auditory stimulation, 60 women; 60 men; Possible explanation: Difference in
finger temperature as a music; skin young students vascular autonomic sensitivity to
response to musical stimulus temperature, index music
60 Men: More asymmetry between Auditory stimulus 15 women; 15 men; Possible hemispheric differences in
hands, larger skin Magnitude and 19–27 y; right-handed response to auditory stimuli
conductance responses on frequency of skin
the left hand conductance
Women: Less asymmetry, responses
larger skin conductance
responses on right hand
65 Acute muscle or skin pain: Skin Skin sympathetic Awake human subjects
blood flow increased in nerve activity
women, whereas in men it Hypertonic saline
decreased injected into tibialis
anterior muscle or
into skin
Skin blood flow
(b) No significant differences
54 Physiological responses to Heat stress; 12 women; 12 men; Differences between women and
heat stress differ with gender, ergometer; oxygen 20–28 y men disappeared when
but depend on fat content uptake; body and differences in the percentage of
and body surface area skin temperature; fat in the body and the ratio
sweat rate between body surface and mass
were taken into account

the indirect response to cold. Thus, sympathetic neural reactiv- Thermal Response to Stimulation
ity, as assessed by way of an indirect response to a cold stimulus, The decrease in finger temperature as a response to musical
significantly contributes to gender differences in the response to stimulus was greater in women (59). This may be due to dif-
local cooling. In contrast, the variation in microvascular respon- ferences between men and women in vascular autonomic sen-
siveness to cold exposure due to the menstrual cycle is most sitivity to music, or to differences in sensitivity or density of
probably caused by local vascular mechanisms rather than by peripheral vascular adrenergic receptors.
variation in sympathetic neural reactivity to local cooling. Electrodermal responses: electrodermal asymmetry has
Sex-related differences in thermoregulatory responses been considered as an index of hemispheric specialization.
while wearing protective clothing were found (58). Women A study recorded the magnitude and frequency of the skin
were at a thermoregulatory disadvantage compared with men conductance responses when subjects listened to tones (60).
when wearing protective clothing and exercising in a hot envi- Subjects were right-handed in order to control the effects
ronment. This disadvantage can be attributed to the lower of handedness. Men displayed more asymmetry between
specific heat of adipose versus non-adipose tissue and higher hands, with larger skin conductance responses on the left
percentage body fatness. hand. In women, asymmetry was less marked, and larger skin

conductance responses were found on the right hand. These the number of melanocytes in both exposed and unexposed
results indicate a possible hemispheric difference in response skin. Another study assessed skin color in adolescents (71). The
to auditory stimuli. forehead (sun-exposed) pigmentation of boys was darker than
that of girls. But the medial upper arm (less sun exposure) pig-
Thermal and Pain Sensation, mentation varied among the different phases of adolescence:
Pressure Sensitivity girls were darker than boys during early adolescence, during
Sensation in the skin can be studied in relation to pain. Pain middle adolescence the pigmentation was similar in the two
can be induced mechanically, electrically, by chemical stimulus sexes, and during late adolescence girls were significantly
or by thermal stimulus. Pain sensation is best determined by lighter than boys.
the threshold at which pain begins, and the stimulus required The lighter skin color of women was attributed to differ-
to produce it can be quantified. Thermal and pain sensations ences in melanin, hemoglobin (variations in vascularity) and
are mediated by cutaneous receptors and travel through carotene (72). Natural selection might give an explanation of
myelinated (Aδ) and unmyelinated (C) nerve fibers. Women the overall visual effect of lighter skin. In addition, women are
were more sensitive to small temperature changes and to pain more homogenous in color than men, since regional variations
caused by either heat or cold (61). Another study measured in reflectance spectrophotomery were smaller in women than
the threshold of the pricking sensation provoked by heat pro- in men (72). Colorimetric measurements revealed a darker and
jected to the skin from a lamp (62). The pricking pain threshold redder skin in elderly men (65–88 years) compared with elderly
increased with age in both sexes. In addition, the threshold of women, but such differences were not found in young subjects
women was lower at all ages in the range 18–90 years. Possible (18–26 years) (73). Another study of 461 women and 346 men
explanations to the difference between the sexes are: aged 20–69 years found that both sexes darken with age (69).
Yet another study did not find differences between men and
• Anatomical differences in skin thickness women in epidermal melanocyte counts (74).
• Differences in blood flow and blood vessels that absorb
part of the heat transmitted to the skin
• Differences in nervous structure or function HORMONAL INFLUENCE (TABLE 1.9)
Unlike the forearm lower pricking pain sensation threshold in Any of the above differences between women and men might
women, pressure threshold was lower in wteomen than men be related to hormonal effects. Some evidence for hormonal
on the palm and on the sole, but not on the forearm (63). influence on the skin has already been mentioned above, like
the increase of skin thickness following conjugated estrogens
treatment of postmenopausal women (9), or the positive effect
Autonomic Function of estrogens on stratum corneum hydration and wrinkles of
Skin conductance measures one aspect of the autonomic the face of perimenopausal women (21), or the changes during
function. Neonate girls manifested a significantly higher the menstrual cycle demonstrated by measuring baseline tran-
conductance than boys (64). These differences may represent sepidermal water loss (41) and skin blood flow (42). Hormone
differences in maturation. replacement therapy for menopause had an effect on skin
Both acute muscle and skin pain evoked a measurable extensibility (75): in untreated women a steep increase in skin
sympathetic activity in human subjects who were awake. extensibility was evidenced during the menopause. Hormone
Sweat release was increased to the same level in men and replacement treatment limited this age-related increase in skin
in women, but dissimilar changes in skin blood flow were extensibility, thus having a preventive effect on skin slackness.
recorded: skin blood flow increased in women, whereas in Other parameters of skin viscoelasticity were not affected.
men it decreased (65). After menopause the skin becomes thinner, associated with
loss in skin collagen content. Collagen content increased with
SKIN COLOR (TABLE 1.8) hormone replacement therapy by 48% compared with non-
An article by Tegner (66) gives several examples of artists treated subjects (76). Moreover, the ratio of type III to type I col-
depicting their female models as lighter skinned than males. lagen in the skin is reduced with age. Postmenopausal women
Such differences were indeed found utilizing spectrophoto- receiving hormone replacement therapy showed an increased
metric measurements, in various ethnic populations. A lighter proportion of type III collagen in the skin (77). In the future,
skin in women was demonstrated in studies from Iran (67), further hormonal manipulation might change the skin of both
India (68), and Australia (69). In addition to hormonal influ- men and women in ways we cannot yet predict.
ences, differences in melanin, hemoglobin, and carotene might
be involved, as well as differences in sun exposure. Skin reflec-
tance spectroscopy was measured in 10 anatomical sites in 20 PILOSEBACEOUS UNIT (TABLE 1.10)
healthy Caucasian babies (mean age 5 months, range 1 to 10 The sebaceous glands are hormone-dependent. The increase in
months). The level of skin pigmentation was the same in all their activity during puberty can be stimulated by the admin-
the 10 measured sites and there were no gender differences in istration of the appropriate hormone. Androgenic steroids,
pigmentation for any site (70). In general, both sexes darken of either gonadal or adrenal origin, have a direct stimulatory
as age increases (69). But the changes are more intricate (68): effect on sebaceous gland activity. Most of the hormones (TSH,
from the end of infancy to the onset of puberty there is a pro- ACTH, FSH, LH) act indirectly by stimulating their respective
gressive skin darkening in both sexes. During adolescence endocrine tissues. In other cases the hormones (for instance
they both lighten, but women lighten more. Simple hormonal GH) act synergistically with another hormone to which the
effects cannot explain this difference, since both testosterone sebaceous gland is sensitive. Average values for sebum secre-
and estrogen provoke darkening rather than lightening of tion were significantly higher in men than in women for age
the skin. These changes might be partly attributed to differ- ranges 20 to over 69, but not for 15–19 years (78). This differ-
ences in exposure to sunlight, since UV irradiation increases ence in sebaceous gland activity becomes more apparent in the

Table 1.8 Skin color

Ref. Finding Obtained by Subjects Conclusions
(a) Significant differences
19 Women’s skin lighter Spectrophotometry Review article Not a simple hormonal effect
Differences in melanin,
hemoglobin and carotene
67 Women’s skin lighter Spectrophotometry 33 women; 68 men; Differential tanning;
8–24 y vascularity variations
68 Women’s skin lighter Spectrophotometry; 566 women; 578 men; During puberty, males darken,
upper inner arm 1–50 y females lighten
Different levels of MSH
Hereditary and
environmental factors
71 Forehead: Boys darker than girls. Skin color, measured by 105 women, 10–16 y; Physiologic changes during
Medial upper arm: Girls darker than reflectance of 105 men, 12–18 y adolescence may cause
boys during early adolescence, not forehead and medial these sex differences
different from boys during middle upper arm, in
adolescence, and during late adolescents
adolescence girls lighter than boys
69 Women’s skin lighter Spectrophotometry; 461 women; 346 men; Different levels of MSH
Both sexes darken with age inner upper arms, 20–69 y Difference in sun exposure
lateral forearms, back (tanning and thickening of
of hands skin)
73 In the elderly: Skin of men darker and Colorimetric 8 women, 5 men;
redder compared with women, but measurements of 65–88 y
not in the young forehead (sun- 9 women, 4 men;
exposed) and forearm 18–26 y
(b) No significant differences
74 No difference between men and 5 mm paraffin 38 skin samples of men
women in epidermal melanocytes embedded sections and women of
counts different ages
DOPA reagent.
73 In Caucasian babies: Pigmentation Colorimetric 10 women, 10 men;
same for men and women measurements of 10 1–10 mo

Table 1.9 Hormonal influence

Ref. Finding Obtained by Subjects Conclusions
Significant differences
75 Hormone replacement treatment Computerized suction Women: 43 nonmenopausal Hormone replacement
limited the age-related increase in device measuring (19–50 y) therapy has a
skin extensibility skin deformability and 25 menopausal not treated preventive effect on
Other parameters of skin viscoelasticity; inner (46–76 y) skin slackness
viscoelasticity were not affected forearm 46 on hormone replacement
therapy since onset of
menopause (38–73 y)
76 Collagen content increased by 48% Hydroxyproline and Postmenopausal women Estrogen or testosterone,
with hormone replacement collagen content; (35–62 y) or both, prevent the
therapy compared with nontreated biopsies of right thigh 29 untreated; 26 estradiol+ decrease in skin
subjects below the greater testosterone collagen content that
trochanter occurs with aging
77 Increased proportion of type III Analysis of collagen Postmenopausal women The clinical improvement
collagen in the skin of types; biopsies of (41–66 y) in the skin following
postmenopausal women receiving lateral thigh 14 untreated; 11 estradiol + hormone replacement
hormone replacement therapy testosterone therapy is due not only
to increase in total
collagen but also to
changes in the ratio of
type III to type I

Table 1.10 Pilosebaceous unit

Ref. Finding Obtained by Subjects Conclusions
Significant differences
79 During January women’s hair was Phototrichogram; hair 7 women, 29–49 y;
denser and the percentage of count after washing 7 men, 25–47 y
telogen hair lower compared with
78 Higher sebum secretion in men than Sebum production 330 women; 458 men;
in women for age ranges 20 to 15 y to over 69 y
over 69, but not for the 15–19 age
In the 50–70 age range the
secretion in men remains
unaltered, whereas in women
there is a significant decrease in
sebum output, probably as a result
of decreased ovarian activity
78 No correlation between sebum Sebum production and 8 women; 28 men
production and plasma plasma androgen
testosterone levels

50–70 age range, when the secretion in men remains unaltered hair follicles was studied in vitro. The distribution pattern of
whereas in women there is a significant decrease in sebum out- ERbeta and TGF-beta2-immunoreactivity differed between
put, probably a result of decreased ovarian activity. male and female hair follicles after 48 h culture. Of 1300 genes
Beginning in young adulthood there is an age-related tested, several genes were regulated differently as relates to
decline in wax ester secretion—thus hormones also affect the gender. Thus, substantial sex-dependent differences were
composition of sebum. found in the response of frontotemporal human scalp hair fol-
The distribution of hair over the body differs between licles to E2 (84).
men and women. The hair follicles possess individual mech-
anisms controlling the evolution and triggering of successive
phases, but systemic factors like hormones and external fac- CONCLUSIONS
tors also play a significant part. The season of the year has Maintaining skin health is an intricate orchestration of many
an effect on hair growth and hair shedding. From data given variables. The need for hard data is paramount, not only for
in a study concerning this seasonal effect (79), we calculated gaining knowledge about the anatomy and biology of human
sex differences, which were not discussed in the study. The skin, but also for the assessment of pathophysiological pro-
data referred to the month of January. Women’s hair was cesses and for clinical management of skin diseases. New and
denser and the percentage of telogen hair lower compared improved instrumentation will allow for more studies, leading
with men. to a detailed description of physiological differences between
The diversity of male and female hair patterns is deter- men and women.
mined by a difference in the transformation of vellus to ter- We hope that this chapter will trigger further investiga-
minal hair, stimulated by androgens, but also by racial and tions of the subject.
genetic factors. In Koreans, women had a significantly higher
number of terminal hairs than men (80).
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Climatic Influence on Cosmetic Skin Parameters

Mathias Rohr and Andreas Schrader

INTRODUCTION between values of about −10 and 25 °C in a year. Relative

In addition to good compatibility, which should be a matter humidity is about 50% in summer and 90% in winter.
of course for cosmetic products, the physiologic effective-
ness, in particular moisture and smoothing effects on the
skin, is the main interest for cosmetic products. Techniques
Positive and Negative Standards
Tests have been carried out with the same products repeatedly
such as fast optical in vivo topometry of human skin (FOITS)
over a period of several years, and these will serve to demon-
(1,2) and corneometry are used to investigate their effec-
strate the effect of climatic conditions on skin physiology. The
tiveness. A high degree of standardization is required to
positive standard is a well-accepted former brand product that
quantify the effects of cosmetics (3,4). To obtain reproduc-
is currently unavailable on the European market. However, we
ible and statistically significant results, experimental condi-
have been making it at a constant quality level for years using
tions, such as test panel–controlled climatic conditions and
the known formulation. This product, referred to hereafter as
a test design including a positive and a negative standard,
“standard L” (Table 2.1), is tolerated very well by the skin and
are the basic starting tools. Nevertheless, as the following
demonstrates a moisture-retaining and skin-smoothing effect
discussion will show, it is not only the normal standardiza-
that can be easily classified in terms of physiologic effective-
tion procedures, such as acclimatization of volunteers in spe-
ness. This makes it an ideal standard, because other products
cial air-conditioned laboratories, which have to be taken into
can be classified as better or worse with respect to their effec-
consideration when interpreting objective and subjective
tiveness. Another aspect of demonstrating the effectiveness of
cosmetic parameters, but also the effect of the actual climate
products on skin physiology relates to negative effects that,
during the application phase and especially during the days
for instance, can be induced by aggressive surfactants. Here,
of measurement. The influence of the indoor climate in the
too, we have been using the same standard product for years.
laboratory as well as the outdoor climate will be analyzed.
This is sodium dodecyl sulfate (SDS), which is referred to as the
What will happen to the level of skin moisture during the
“negative standard” from now on.
preconditioning phase or what will happen at different sea-
sons of the year? Will it be influenced by the level of relative
room humidity and/or the actual climate conditions? Will Laser Profilometry
the influence vary for different kinds of products? Will the The laser profilometry technique is used to investigate the anti-
influence on skin moisture and skin structure be compara- wrinkle effect. Skin replicas are taken from the test areas on
ble? Will the influence change for different types of volun- the volar forearms by means of a white pigmented silicone sub-
teers? What is the best time for preconditioning? Could the stance (two components, Optosil, Bayer, Inc., Germany), before
regeneration of the stratum corneum be influenced by the the first application and 12 hours after the last application. A
climate? Will effects felt subjectively (washing the bend of round impression having a diameter of 18 mm is made using
the elbow) be equally dependent on climatic conditions as a label especially designed for this purpose. While the impres-
objectively rated parameters? sions are being made the volunteers are seated on chairs with
A summary of individual results and averages of thou- adjustable armrests so that the angle between the upper arm
sands of volunteers will be given. Both a positive standard (in and the forearm can be adjusted to 90°. Fixing the forearms
the sense of increasing moisture and smoothness) and a nega- in this way ensures that no factitious smoothing or roughen-
tive standard (in the sense of increasing dehydration, rough- ing effects, due to stretching of the arms when the impressions
ness or side effects) are used to present the effect of climatic are taken after application, are evaluated and included in the
conditions on skin physiology tests. documentation.
An automated laser scanner with an optical autofo-
cus sensor is used for contactless scanning of the skin repli-
MATERIALS AND METHODS cas (UBM, optical measuring system Microfocus, UBM RC14,
Climatic Data Karlsruhe, Germany) (5). The measuring range of the laser
To be able to correlate climate data with skin physiology scanner is ±500 mm at a resolution less than 0.01% of the mea-
parameters, the relative humidity and outside temperature suring range. The measuring spot (focus of the laser diode)
are measured continuously at a station by a computer (CAN has a diameter of about 1 mm. The z resolution is increased to
system, Lufft Company, Fellbach, Germany). Capturing the ±25 mm by an additional shift of the z-axis if necessary. The
data by computer ensures that the climate is recorded day and resolution in the x- and y-directions is identical to be inde-
night. Let us take climatic changes in Holzminden (longitude pendent of any predominant direction of wrinkles. The skin
9.27 east and latitude 51.49 north; Middle Germany) over a year replica taken from the volar forearm of a volunteer is scanned
as an example. As Figure 2.1 shows, temperature fluctuates over an area of 8 mm × 8 mm in the x- and y-directions at a

100 Ra/DIN 4768/1

Temperature (ºC) relative humidity (%)

80 Ra


15 Yi: Absolute value of the profile
5 Ra = 1/n Σ |Yi|
Aug Nov Feb May Aug
Rz/DIN 4768/1
Figure 2.1 Climatic outdoor conditions at Holzminden, Germany, Y1 Y2 Y3 Y4 Y5
from August 2001 to 2002.

Table 2.1 Declaration of Positive “Standard L” According to the
International Nomenclature of Cosmetic Ingredients
Water Yi: Maximum profile difference (Rmax)
Liquid paraffin 5
Caprylic/capric triglyceride Rz = 1/5 Σ |Yi|
Hydrogenated coco-glycerides (b)
Myristyl alcohol
Figure 2.2 Definition of DIN parameters Ra (a) and Rz
(b) according to DIN 4768/1.
Glyceryl stearate
Cetyl alcohol
Proprietary composition
4-Methylbenzylidene camphor
Tocopheryl acetate
of these five individual values. The Rz parameter will indicate
Butyl methoxydibenzoylmethane roughening of the skin profile by a significantly increased
Aloe barbadensis value if the profile is changed by the influence of a product
Isopropyl myristate (Figure 2.2).
Polyaminopropyl biguanide
Soluble collagen OF HUMAN SKIN
Simethicone After a successful validation phase, the new FOITS technology
Sodium hydroxide was introduced in 1997 (1). In comparison to the replica-driven
Ethylenediaminetetraacetic acid
technique during the previous decade, the touch-free tech-
nique of fringe projection became state-of-the-art to investigate
skin surface (2,9–11). Because of many technical advancements
resolution of 25 points/mm. Thus 40,000 individual measure- (for example, improved camera resolution, the use of blue LED
ments are available, permitting an exact three-dimensional lighting systems, or laser-supported and computer-optimized
reconstruction of the skin surface (5,6). overlaying procedures), an easy-to-operate system has been
realized recently. As there has always been a great deal of sci-
Ra Parameter entific interest on the mechanisms of wrinkle evaluation, the
The Deutsche Industrie Norm (DIN) parameter Ra represents technical developments led to a tool of high scientific standard
the mean roughness index according to DIN 4768. Ra indicates (12–15).
the arithmetic mean of the absolute values of the skin profile’s FOITS is a touch-free optical technique with a history
deviations from the center line over the total distance. of more than a decade of investigating skin surface struc-
If the overall structure of the profile remains unchanged tures in a direct three-dimensional measurement by fringe
(Rz constant) but the fine structure of the profile changes, then projection (16). The fringe-projection technique used is a
the Ra parameter will indicate smoothing or roughening by a combination of gray-code and phase-shift technique (7). In
reduced or increased value, respectively (7,8). less than a few hundred milliseconds, the absolute space
coordinates of all object points in the selected image area
Rz Parameter are measured with great precision. The FOITS measurement
The Rz parameter represents a mean peak-to-valley height system consists of a projection unit and a CCD camera. Both
according to DIN 4768/1. If, in the two-dimensional case, a pro- are fixed under the triangulation angle. In the gray-code
file line is divided into five equal parts and the Rmax param- method, grids with a rectangular brightness distribution
eter is calculated for each part, Rz will be the arithmetic mean by different numbers of lines are projected. The number of

lines is doubled at each new projection. This gives a clearly of the CCD camera. The high resolution in the vertical direc-
defined hierarchy of lines for each image point. In the phase- tion is achieved by analysis of the intensity and phase dis-
shift technique, only one grid with a sinus-like intensity placement of the projected grids. The surface structure of the
distribution is projected several times with different phase analyzed area causes a deviation of the intensity and phase
positions. The FOITS technique is able to realize a depth information of the projected grid structures from the theo-
sharpness area of ±10 mm on an inspection area of 30 mm × retical model structure of a plane surface. With correspond-
40 mm. The resolution in the vertical z-direction with 0.2% of ing mathematical algorithms, the absolute three-dimensional
the measured area leads to an effective resolution of 4 mm in coordinates of the inspected area can be calculated of these
the z-direction. A CCD camera with horizontal and vertical deviations. A synopsis of the most important experimental
resolution in x- and y-directions of about 30 mm is used. The side parameters is shown in Figure 2.3, from the first experi-
resolution in the z-direction is not limited by 256 gray steps ments up to the current time (Figure 2.4).

FOITS 1995 1998 2003 2006

Gray-code and phase-shift technique
Technique Contact free direct skin measurement in vivo
Halogen light Blue LED technique
Superimposition Mechanically aided by online overlay procedure LASER aided Software aided on top
mechanically of all
Measurement area Inner side of the forearm Crow’s-feet, under the eye, cheek, glabella, lips, nasolabial, dé colleté,
forearm, leg
Area of inspection 875 mm2 (25 mm × 35 mm) 1200 mm2 (30 mm × 40 mm)
Area of analysis 20 mm × 20 mm 20 mm × 20 mm (or as needed)
Resolution x-direction ~40 mm ~30 mm
y-direction ~40 mm ~30 mm
z-direction 4 mm 4 mm
Time to digitize the fine ~320 msec ~260 msec

Figure 2.3 Synopsis of the Technical Side Parameters of FOITS.





Touch Free
Real Time
3D-Analysis Of Skin Surface
Fringe Projection

End value

Figure 2.4 Presentation of various FOITS system from 1995 to today; example of FOITS data presentation on an individual subject.
3-DIM data presentation of the crow’s-feet area before and after 4 weeks of product application.

Starting with analysis of the inner side of the forearm, the area of analysis into these five subareas (areas 1 to 5, see
the crow’s-feet area eventually became the area of most interest. Figure 2.5), the area close to the eye, called area 1, represents
Increasing the power of FOITS technique as described in Figure the deepest structures, while with area 5 smaller structures are
2.3, more areas could be investigated such as the cheek, glabella quantified. An example of this analysis is given in Figure 2.4.
area, under the eye, nasolabial area, lips, or all body areas such In comparison, analysis of the lip area is shown. Because of the
as the décolleté and legs. The latest technique combines the fast- smaller test area, only four areas are defined with 40 separate
est data measurement with the best superimposition technique lines with a distance of 250 mm. As shown by Figure 2.1, cor-
to guarantee a perfect comparison of baseline and end-value relation of line number and Rz results in a more flat link for the
data. Superimposition is realized in a combination of laser- lip area in comparison to the crow’s-feet area.
aided mechanical alignment of the subject in a first step fol- To document the surface structure by a global parameter,
lowed by a software-driven rotation and shifting procedure of the frequency distribution of all depths is used. The FDD is cal-
measured data/pictures to find the optimum superimposition. culated in the range from −600 mm to 600 mm (after polynomial
correction) by using interval steps of 5 mm. The defined evalu-
Parameter of Analysis ation area is equivalent to a surface of 2 cm × 2 cm and accord-
Bringing into focus the periorbital wrinkle area (crow’s-feet), ing to the technical resolution of the camera represents 640,000
the morphological structure of this test area has to be taken single points. Therefore, a calculated FDD parameter is based on
into account if wrinkles are investigated. Having this in mind, a rearrangement within these 640,000 values of depth.
analysis is carried out perpendicular to the main wrinkle Working with a distribution function, the zero level has to
direction based on the Rz parameter (according to DIN 4668 be kept in mind. Thus, the zero level of each volunteer is defined
[12]) or the frequency distribution of depth (FDD) analysis. as the first plane representing a level of about 0.1% of all single
Starting close to the eye, 50 separate lines with a distance of values (about 600 counts). This plane is set as zero and all further
400 mm are analyzed. The resulting roughness is shown as calculations are done with these resulting standardized values.
a function of line number (Figure 2.5). Ten successive lines From the surface structure, a frequency distribution of all depths
are averaged, resulting in five areas of evaluation. Separating is obtained, as shown exemplarily in Figure 2.6 (left curve).

Area 1 2 3 4 5



Rz [mm]


Crow’s feet
Area 1 Area 2 Area 3 Area 4 Area 5
0 10 20 30 40 50
Line number

Figure 2.5 Definition of subarea of analysis. Rz as a function of subarea lines of an individual example in the crow’s-feet area and
lip area.

Table 2.2 Summary of Experimental Conditions for the Various

Skin Physiology Tests
Investigation brief description corneometer 20–30 volunteers
Improvement Baseline
2–3 wk of application; twice a day
End value
Baseline measurement on the forearm
Final value 12 h after the last application
20000 Statistical analysis of data
Corneometer kinetic frequent measurements up to 5 h

15000 Laser profilometry 30 volunteers

3 wk of application; twice a day
Silicone replica of the forearm (baseline)
10000 Rough structure >–170 µm Silicone replica 12 h after the last application (final value)
Fine structure –55 to –170 µm Robot-controlled laser profilometry
5000 Microstructure 0 to –50 µm Analysis of Ra and Rz
FOITS frequent measurements up to 4 h
No replica
0 Analysis of Ra and Rz
0.0 0.1 0.2 0.3 0.4 0.5 0.6
Washing test on the bend of the elbow 20 volunteers 5 days of
Depth [mm] application
Twice a day, 2 × 1 min of washing
Figure 2.6 Histogram of depth of a surface profile (crow’s-feet Subjective rating of side effects in a direct comparison
area), classification of structural regions as well as visualization Reddening/stinging/skin tautness/itchiness
of smoothing effect/age effect–baseline: 65-year-old subject, end Skin roughness/dull feeling/bad skin feeling
value: 15-year-old subject. Statistical analysis of reaction points
DHA decoloring 20 volunteers, aged >50 years
Measurement of skin color by chromameter (baseline)
Application of DHA to inner side of forearm
According to the selected zero level, a classification of Application of test product twice a day for 18 days
Measurement of skin color every day
depth is made as follows:
Analysis of decay curves
• 0 to 50 mm → Microstructure (about 5%)
• 55 to 170 mm → Fine structure (about 65%) Abbreviation: DHA, dihydroxyacetone.
• <170 mm → Rough structure (about 30%)
The given proportion will give a rough estimation of All tests mentioned in this discussion were carried out
structure ranges found in the crow’s-feet area of women with in an electronically controlled air-conditioned laboratory that
distinct wrinkles and Caucasian skin. Taking into account a ensures that room temperature and air humidity are kept
product’s smoothing effect, the green FDD curve as shown constant. The volunteers were kept seated in this laboratory
in Figure 2.4 can be expected. Consequently, an improve- at 22°C (±1) and 60% or 50% (±5%) relative humidity for 45
ment of skin structure is defined by a shift of maximum and minutes before the test and during the complete standard test
a change of width of the distribution function. A reduction of procedure.
rough structures can be expected, while for fine- and micro- To quantify the influence of this procedure of standard-
structures an increase is obtained in the case of structural ization, frequent measurements were carried out immediately
improvements. after the volunteers arrived at the institute and for up to 5 hours.
To show the basic influence of the indoor climate, no product
Corneometer application was performed during the time of the investiga-
Water differs markedly from most substances as far as its dielec- tion. In a second series of measurements, five different brands
tric constant is concerned. A quantitative proof of changes to and five different formulations with an increasing amount of
the water content of the skin can thus be achieved in a noninva- glycerine (3%–25%) as an active ingredient were investigated
sive manner by means of capacity measurements (17,18). in a short time test design up to 4 hours after product appli-
A corneometer (Courage + Khazaka Co., Köln, Germany) cation. To quantify the influence of the indoor climate on the
is used to measure the water content (Table 2.2). A measuring product rating, the second test series was carried out twice. In
capacitor reacts to the samples in the volume to be measured a first run, the relative humidity was set at 60%; in a second
by way of capacitance changes (depending on water content). run the relative humidity was reduced to 50%.
Those capacitance changes registered by the measuring head Transient individual side effects that may have an influ-
capacitor are processed fully automatically by the equip- ence on the skin are standardized in this way. However, this
ment to form a digital measured value. There is no conduc- procedure does not compensate for climatic conditions such as
tive (galvanic) connection between the object measured and winter or summer.
the measuring equipment. Consequently, almost no electricity
flows through the object measured. Properties such as ionic Regeneration
conductivity and polarization effects have no influence on Dihydroxyacetone (DHA) is a substance that is tolerated very
the measurement result. The fact that the electronics adapt well and is approved in the cosmetics industry as a suntan sub-
to  the  moisture circumstances almost without inertia means stance. It tans by means of the Maillard reaction, forming com-
that the measuring process is very fast and that it is possible, to binations with amino acids in the skin that do not wash off.
a considerable extent, to eliminate effects on the results caused The color disappears within approximately 3 weeks as a result
by involuntary movements or moisture accumulation during of desquamation of the colored horny cells. The tan of the skin
the measuring process. decreases accordingly.

For this investigation the desquamation effect, and con-

sequently the rate of regeneration, is measured in the labora-
tory color room by measuring the decoloring with a Minolta 14 Standard L
Chromameter CR 300 (L-a-b color room). The yellow value b
differentiates best, and this is used to establish the color decay
curves (19,20).

Δ Corneometer (%)
The region that is tested is again the volar forearm. Areas
of 4 cm × 4 cm in the middle of the region of application are 8
colored with DHA after a defined washing procedure to stan-
dardize the baseline conditions. In the coloring process, a spe- 6
cial emulsion with 10% DHA is applied to the area to be tested. 4
The amount applied is 6 mg/cm2. In addition, an adhesive
bandage saturated with DHA emulsion is applied for 24 hours. 2
Over the next 18 days, the volunteers continue to use the prod-
ucts twice a day. The forearms are permitted to be washed 0
1 2 3 4 5 6 7 8 9 10 11 12
only twice a day with warm water. Surfactants and abrasive
cleansing agents are not allowed to be used. Measurements Month
are taken directly before DHA coloring, and then every day
Figure 2.7 Percentage increase in moisture, after correction
over the next 18 days with the exception of weekends. For each
for the untreated area, of positive standard L monthly summary
time and area of measurement, three values are recorded at (12 hours after last application, 4460 volunteers, 1992–1999).
different places in the measurement area and averaged. The
b-values of all 30 volunteers per product are averaged, and the
standard deviations, percentage changes, and percentage dif-
ferences standardized to the coloring are calculated. The color
decay curves can be described under normal conditions with 40
the following exponential function: Standard L
Δ Corneometer (%)

b = a1e − a2t + a3
Further statistical treatment is described in detail in Refs. 3 and 9.
Washing Test on the Bend of the Elbow
To assess the skin tolerance, the cleansing effect, and the –40
acceptance of surfactant products, we carry out the washing
test on the bend of the elbow. In a practical test, the bend of –60
the elbow is washed under intensive conditions. Twenty vol- 0 1 2 3 4 5 6 7 8 9 10 11 12
unteers take part in this test. In each application, the bend of Month
one elbow is lathered vigorously with the first sample and
washed for 2 minutes by hand. After being rinsed with luke- Figure 2.8 Standardized differences of moisture for the positive
warm water, this bend of the elbow is again lathered and standard L after correction for the untreated area (12 hours after
the last application, 3100 volunteers, 1992–1995).
washed for 2 minutes. This is followed by a period of drying
also lasting 2 minutes. After the second rinsing with luke-
warm water, the area is carefully dabbed dry with a towel,
ensuring that there is no rubbing. The bend of the other Calculations led to an average moisture increase of
elbow is treated in exactly the same way with the negative approximately 12.7% for all data recorded. To make it easier
standard SDS (21,22). to compare seasonal dependency of the achievable moisture
To determine any side effects induced by the test prod- increase, Figure 2.8 shows the difference from the overall aver-
ucts, the volunteers are asked at the end of the test about any age after the data have been standardized on the basis of the
reactions they noticed directly after washing. The following overall average. A change of 0% corresponds to the above-
parameters are ascertained: reddening, stinging, skin taut- mentioned overall average of approximately 12.7% moisture
ness, itchiness, skin roughness, dull feeling, and dehydrated increase. A bar in the positive direction thus shows an increase
skin feeling. The ratings are given on the basis of a coded vol- in moisture that is higher than the average, whereas a bar in the
unteer questionnaire. negative direction indicates a reduced level of effectiveness.
Figure 2.8 shows that from November to February, there was
RESULTS AND DISCUSSION about 15% above the average moisture increase, whereas in the
Outdoor Climate summer months of June, July, and August, the level of effec-
One of the major factors in cosmetic skin physiology is the tiveness was approximately 50% below the average achievable
moisture-retaining effect of a product. Figure 2.7 shows moisture increase.
a summary of this for 1992–1995. The data have been sum- Figure 2.9 shows the relative change of the laser profilom-
marized on a monthly basis in each case. The percentage etry parameters Ra and Rz both for the positive standard L and
increase in moisture induced by the positive standard L after for the untreated area in a way that is comparable to Figure
correction for changes in the corresponding untreated area is 2.7. The area referred to as “untreated” has not been treated
shown. The recorded averages are based on at least 100 volun- with a cosmetic but has been subjected to a washing proce-
teers a month. dure to obtain better results, as described in the “Materials and

Δ Ra/RzDIN (%) 0


Ra standard L Ra untreated
RzDIN standard L RzDIN untreated
0 1 2 3 4 5 6 7 8 9 10

Figure 2.9 Percentage of differences for the DIN parameters Ra and Rz for the positive standard L and the untreated area in a summary
of laser profilometry data (1000 volunteers in general, 12 hours after the last application, 1994–1996).

Δ Ra/RzDIN (%)



0 1 2 3 4 5 6 7 8 9 10

Figure 2.10 Differences of the DIN parameters Ra and RzDIN after correction for the untreated area in laser profilometry (12 hours after
last application, 1994–1996).

Methods” section below. Figure 2.9 shows clearly how impor- level of the skin is increased in the summer months to such an
tant this prior treatment is. Whereas the Ra and Rz parameters extent that, first, skin moisture and smoothing can be increased
for the positive standard fluctuate between −6% and −8% from further by cosmetics to only a limited degree and, second, that
January to October 1994 to 1996 without showing a definite the deliberate use of substances that are detrimental to the skin
trend, these parameters fall noticeably for the untreated area also has a limited negative effect. This leads to an apparent
from January to August, followed by a rise in September and reduction of cosmetic effectiveness.
October. After allowing for the untreated area, the profilom- In addition to these objective skin physiology param-
etry tests result in the dependency that is shown in Figure 2.10. eters, subjective information gained from volunteers’ answers
Again, the positive standard L was found to be less effective on to questions indicates a comparable dependency on external
average in the summer months of June, July, and August than climatic conditions. Figure 2.11 shows the total negative reac-
in the other months. tion points that volunteers gave for reddening, stinging, skin
The data clearly show that the seasonal dependency was tension, itchiness, skin roughness, dull feeling, and bad skin
based on both the reduced positive effectiveness of standard feeling in the elbow washing test. The negative reaction points
L in the summer and the reduced negative sensitivity of the for the negative standard fluctuated between 11 and 18 in May,
untreated area (prior treatment with a surfactant of all areas depending on the comparative product. Since the comparative
tested). External climatic conditions thus have a distinct influ- product is of crucial importance in rating effects subjectively,
ence on the cosmetic effects that can be achieved. The basic the same test setup was repeated in November with the same

comparative products. Here, the average total negative reaction there will be a constant exponential reduction of skin color-
points for the comparative product SDS were distinctly higher ing of both the untreated area and the areas that have been
in all four groups taking part in the test. Whereas the average treated with the test products (19). Figure 2.12 shows average
for May was approximately 15 negative reaction points, this curves that have been standardized to the maximum coloring,
rose to approximately 23 reaction points in November under on the basis of 20 volunteers for two test products (A and B)
otherwise identical conditions as far as the volunteers’ subjec- containing α-hydroxy acids and one untreated area. The obser-
tive feelings were concerned. These data, based on 80 volun- vation period was 18 days. In contrast to theoretical expecta-
teers, clearly show that it is possible and necessary to correlate tions and preliminary experiments, this investigation revealed
information derived from volunteers’ subjective ratings with a reduction in skin coloring from about 70% to about 30% on
climatic conditions and to consider this along with the objec- day 8. Both before and after this sudden change, the curve is in
tively demonstrable parameters for skin physiology. keeping with theoretical expectations. When all potential tech-
Another example of how external climatic conditions nical sources of error had been eliminated, the solution to this
make it almost impossible to evaluate the results of skin physi- problem was found in the temperature and relative humidity
ology investigations is the turnover of the stratum corneum on data for the days of measurement, as shown in Figure 2.13.
the basis of DHA decoloring tests. When the stratum corneum As the curves show, relative humidity fell from about 90% to
has been colored with DHA, it can generally be expected that about 60%, whereas the temperature rose from about 0 to 68C
over the same period of just a few hours, and then fell to 18C
after a short time. Since temperature/humidity fluctuations
were far less extreme in the rest of the test period, it seems
SDS, May reasonable to suppose that the strong fluctuations of tempera-
25 SDS, November ture and humidity correlate with the recorded inconsistency
in the DHA color decay curves. This inconsistency induced by
Negative reaction points

20 extreme climatic fluctuations made it necessary to repeat the

test, because it was no longer possible to carry out an exponen-
15 tial analysis of the decay curves.
As the measured curve was constant before and after
10 day 8 but higher humidity fluctuations accompanied by lower
temperature fluctuations were recorded on day 7, it can be
5 assumed that humidity is of greater importance in examining
the regeneration of the stratum corneum and that the outside
0 temperature plays only a subordinate part in the quality of this
1 2 3 4
Comparison groups skin physiology investigation.

Figure 2.11 Negative reaction points in a subjective rating Indoor Climate

system for four individual comparisons of the negative stan- Figure 2.14a presents the results of the “no-product corne-
dard sodium dodecyl sulfate (SDS) to four different products in ometer kinetic” (i.e., without application of a product). The
a washing test on the bend of the elbow (20 volunteers in each kinetic measurements were carried out on four different test
comparison). areas (forearm—lower, middle, and upper—and upper arm).
In Figure 2.14b, the forearm data are summarized on the basis

Product B
Product A
80 Untreated
Δ-b-Color value (%)




0 2 4 6 8 10 12 14 16 18

Figure 2.12 Exponential decay curves of the dihydroxyacetone (DHA) decoloring test standardized to the maximum coloring character-
ized by changing of the b-value of the L-a-b color room.

7 100


Relative humidity (%)

Temperature (ºC)
4 80

2 70


–1 50
6.0 6.5 7.0 7.5 8.0 8.5 9.0

Figure 2.13 Climatic data of temperature (grey) and relative humidity (black) from day 6 to day 8 during the dihydroxyacetone (DHA)


Upper arm
Corneometer units (CU)

Forearm upper


46 Forearm middle

Forearm lower
0 60 120 180 240 300
Time (min)

6 < 40 CU
40–55 CU
>55 CU
Difference from baseline (%)






0 60 120 180 240 300
Time (min)

Figure 2.14 (a) Kinetic corneometer—data summarized for different test areas, without any product application (n = 120). (b) Kinetic
corneometer—difference from baseline; data summarized for different volunteers, without any product application (n = 120).

of the first measured value. The first group had starting val- below 40 CU showed a constant increase of approximately 2%,
ues below 40 corneometer units (CU), the second group sum- whereas for the group with high starting values above 55 CU,
marized the volunteers between 40 and 55 CU, and the third a decrease of up to 10% was obtained. Independent of the test
group was based on starting values above 55 CU. site, the preconditioning phase seems to be most effective for
Analyzing the data of different test areas resulted in a a high skin moisture level at the beginning of the study. A dry
decrease of about 2 CU for the upper forearm and a little less skin might be less influenced by the indoor climate. The data
for the other test areas independent of the absolute level, which to determine the optimal time of preconditioning to generate
was different for each test site (lower forearm < middle fore- stable skin conditions are represented in Figure 2.15.
arm < upper forearm = upper arm). These data were calculated As shown in Figure 2.15a, the difference from baseline
without taking into account the individual skin type of the (–D– curve: mean overall) became stabilized at 30 minutes and
volunteers. Figure 2.14b reflects this, showing the individual remained constant from 60 minutes on. Thus, 45 minutes of
starting conditions. As can be seen from the differences from acclimatization seems to be the best choice—a time not too
baseline, the group with 40 to 55 CU did not show any changes short for “moist” skin and not too long to reflect a reliable test
above about 1% during 5 hours of investigation. The group design.

Difference from baseline (%)




–8 < 40 CU
40–55 CU
–10 >55 CU
0 10 20 30 40 50 60 70 80 90
Time (min)

Difference from baseline (%)



0 60 120 180 240

Time (min)

Figure 2.15 (a) Kinetic corneometer—difference from baseline; data summarized for different volunteers up to 90 minutes, without any
product application (n = 120). (b) Kinetic FOITS and corneometer—difference from baseline mean overall up to 240 minutes, without any
product application (n = 120/40).

The data describing the skin surface are given in Figure be possible, a second run of five formulations with an increas-
2.15b. No significant changes occurred during the 4-hour ing amount of glycerine was carried out under the same condi-
kinetic investigation. Differences between lower and upper tions. In this case, significant changes occurred for the first two
forearm were comparable to the corneometer measurements. low-glycerine concentrations (concentration below saturation).
Summing up the Rz and Ra values for up to 4 hours, no trend At 50% relative humidity, the level of measured absolute units
in the changes was observed. Consequently, the influence of decreased significantly. Thus, the selectivity became better if
the indoor climate seems to be of minor impact if compared the relative humidity was reduced and the product contained
to skin moisture. In any case, changes of the skin structure are hygroscopic active ingredients. The hygroscopic ingredient
obviously on a much slower time scale if the producing event seems to pick up the air humidity like a sponge as long as it
is as indirect as the indoor climate. is in the upper stratum corneum. Nevertheless, the origin of
Changing the kinetic view to more static analysis, the moisture should be irrelevant for the skin, but in the case of
data of five different brands are summarized in Figure 2.16. ranking and differentiating products as quickly as possible
Figure 2.16a shows the difference between baseline and end after the product application, it might be helpful to measure at
value 4 hours after unique product application in absolute CU. 50% relative humidity.
The dark gray bars represent the data at an indoor climate of
60% relative humidity, whereas the light gray bars are obtained CONCLUSION
at 50% relative humidity. The data recorded, from both objective skin physiology param-
With the exception of product no. 1, no difference eters such as moisture and smoothness and subjective factors in
occurred from changing the indoor humidity. For product no. 1, the elbow-washing test, clearly show that such tests are influ-
a tendency was calculated for the comparison of both measure- enced considerably by climatic conditions. Differences, such as
ments. Taking product no. 1 as a hint that an influence might between summer and winter, cannot be compensated for by accli-
matization in air-conditioned laboratories. Alongside standard-
ized measurement conditions, it is therefore essential to record
the quality of the test panel not only by including an untreated
20 area but also by means of a positive or negative control. Only in
60% relative humidity this way is it possible to establish a classification system for test
50% relative humidity products that is not dependent on a particular season and allows
after correction by untreated

15 the quality of cosmetic products to be rated objectively.
td – As demonstrated by the obtained results, the indoor cli-

Difference (CU)

mate also plays an important part in cosmetic efficacy testing.

10 – In addition to the outdoor climate, which might have an effect
on a long-term basis, the indoor climate (especially the time of
preconditioning) is decisive for short-term and kinetic inves-
5 tigations. While the influence of the moisture level is strongly
dependent on the starting value, the changes of the skin
topometry seem to be not so marked. On the basis of the cor-
0 neometer kinetic data, 45 minutes of preconditioning appears
1 2 3 4 5 to be an optimal compromise between effect, standardization,
Brands and costs. The laboratory conditions (relative humidity) may
also be of great influence. Depending on the active ingredients
(hygroscopic or not), a ranking of products might be of greater
25 60% relative humidity – selectivity if a lower level of relative humidity is used.
– The data presented underline the importance of a stan-
50% relative humidity –
after correction by untreated

dardized procedure to investigate cosmetic effects on a statisti-

cal and reproducible level.
Difference – (CU)

20 **

1. Rohr M, Schrader K. Fast optical in vivo topometry of human
** skin (FOITS)—A comparison to LASER-profilometry. 5th
15 Congress of the International Society for Skin Imaging, Vienna,
2. Rohr M, Schrader K. Fast optical in vivo topometry of human
skin (FOITS). SÖFW-J 1998; 124(2):52–59.
10 3. Rohr M, Schrader A. FOITS (fast optical in vivo topometry
1 2 3 4 5 of human skin)—A classical method in modern efficacy test-
Increasing glycerine concentration (3%–25%) ing. A history of fringe projection in cosmetics. SÖFW-J 2009;
(b) Jahrgang; 135(8):2–10.
4. Jaspers S, Hoperman H, Sauermann G, et al., Rapid in vivo mea-
surement of the topography of human skin by active image tri-
Figure 2.16 (a) Corneometer for brands 1–5. Difference from
angulation using a digital micro-mirror device. Skin Res Technol
baseline after correction by untreated. (b) Corneometer for
1999; 5:196–207.
increasing concentration of glycerine (bar 1, 3% increasing to bar
5. Rohr M, Schrader K. Surfactant-induced skin roughness:
5, 25%). Difference from baseline after correction by the untreated
Quantitative analysis of the surface structure of the skin via
test area. **, significant difference; –, no significant difference.
automated non-touch laser scanning. Eurocosmetics 1994;

6. Hartung J. Statistik, Lehr - und Handbuch Der Angewandten 14. Rohr M, Yan Qi, Schrader A. Anti-wrinkle performance of cos-
Statistik. 9th ed. Munich, Oldenburg: Oldenbourg-Verlag, 1993. metic products differentiated by FOITS, a statement of effec-
7. DIN 4768: Ermittlung der Rauheitskenngrössen Ra, tiveness, age and ethnic background, IFSCC Congress 2006;
RzDIN,  Rmax mit elektrischen Tastschnittgeräten: Begri ffe, Japan.
Messbedingungen. Berlin: Beuth Verl, 1990. 15. Rohr M, Brandt M, Schrader A. Skin surface—Claim support by
8. Stout J, Sullivan PJ, Dong WP, et al. The development of meth- FOITS. SÖFW-J 2000; 126(8):2–11.
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Report EUR 15178 EN. Eur Community, 1993. industriellen Praxis. München: Franzis-Verlag GmbH, 1993.
9. Lagarde JM, Rouvrais C, Black D, et al. Skin topography mea- 17. Schrader K. Untersuchungen wasser-retinierender Kosmetika
surement by interference fringe projection: A technical valida- auf der Haut. Parfümerie Kosmet 1981; 62:265–272.
tion. Skin Res Technol 2001; 7:112–121. 18. Piérard GE, Piérard-Franchimont C. Dihydroxyacetone test
10. Jaspers S, Hoperman H, Sauermann G, et al. Rapid in vivo mea- as a substitute for the dansyl chloride test. Dermatology 1993;
surement of the topography of human skin by active image tri- 186:133–137.
angulation using a digital micro-mirror device. Skin Res Technol 19. Rohr M, Schrader A, Schrader K. The multifunctional effects of
1999; 5:196–207. AHA. Parfümerie Kosmet 1996; 77:762–767.
11. Ferraq Y, Black D, Lagarde JM, et al. Use of 3-D imaging tech- 20. Neter J, Wassermann W, Kutner MH. Applied Linear Statistical
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Human Skin)—A classical method in modern efficacy testing—a reizlindernder Stoffe an der menschlichen Haut am Beispiel
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comparison between Chinese and European populations—a gskriterien mit Seifen und Syndets. Parfümerie Kosmet 1990;
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Transepidermal Water Loss

Jan Kottner and Annika Vogt

INTRODUCTION and order of these lipids is considered most important for

The physician Santorio Sanctorius (1561–1636) who lived more water barrier function (8,14) and they are widely considered to
than 500 years ago in Italy is considered to be the first person be the major barriers for perpendicular water transport (6,9,15).
to work systematically on the phenomenon called perspiratio The composition of the intercellular matrix, however, is com-
insensibilis, the invisible loss of water from the human body. plex and includes lipophilic, but also hydrophilic subcompart-
Since then, it was widely agreed that water gets lost via the ments. Although hardly detectable under normal conditions or
respiratory system and via sweating, but alternative passages on tissue sections, studies suggest that such aqueous regions
of water through the skin were less clear (1). In 1911 Loewy and can be expanded to tube-like structures, e.g., by means of
Wechselmann proposed for the first time that besides sweat- sonophoresis, which enable entry of large molecules (16). Such
ing there is the possibility of passive water diffusion through phenomena are further supported by work that focuses on
the skin (2). Why and how this water penetrates and leaves the pathways of deformable carrier systems across intact skin (17).
skin was another issue of debate for the following 40 years (3,4). Finite element and microscopic transport models increas-
Irrespective of these discussions, the American dermatologist ingly support the importance of the transcellular permeation
Stephen Rothman introduced the term transepidermal water loss pathway for hydrophilic chemicals, including water (6,13,15).
(TEWL) in 1955, characterizing continuous water loss through The surface area of the corneocytes is also much larger com-
the epidermis excluding sweat secretion (5). Today TEWL pared to the intercellular lipids and therefore the total amount
plays an important role in characterizing the skin barrier func- of transcellular water transport and the subsequent evapora-
tion in physiological and pathological conditions. This chap- tion are major determinants of TEWL in healthy skin under
ter gives a brief overview about the structural and functional physiological conditions (13).
determinants of TEWL, how it can be measured, and what this
BIOLOGICAL BACKGROUND The flux of water through the SC cannot be observed directly.
Without effective protection of the “moist inside” of the human Today the established method of measuring TEWL is by
body, life in the dry environment on land would be impossi- placing a measurement probe on the skin and recording the
ble. The main anatomical barrier limiting the amount of water amount of water that evaporates from the skin surface. The
loss is in the stratum corneum (SC). At most skin areas the SC measured water flux density is expressed in g/m2/h. In order
consists of 15 to 20 stacked layers of flat, terminally differenti- to infer that this amount of evaporated water per area and
ated keratinocytes connected by desmosomes embedded in a time equals the water flux within the SC several assumptions
matrix of lipid bilayers. Depending on the hydration state the need to be met: two of the most important are that sweat gland
diameter of the corneocytes is approximately 20 to 50 μm and activity is reduced as much as possible, and that the water flux
the thickness is approximately 1 to a maximum of 3 μm if fully within the SC is in a steady state. In clinical and research prac-
hydrated (6,7). The thickness of the intercellular lipid bilayer tice this is accomplished by standardizing the measurement
is up to 0.1 μm (Figure 3.1) (8–10). This paper-thin but dense conditions and procedures, but total control of these variables
and compact structure separates the outermost boundary, the is impossible (18).
skin surface, from the living epidermal cells underneath. From Several TEWL measurement devices are commercially
the deeper highly hydrated tissue there is a constant flux of available. They can be classified as open-chamber and closed-
water molecules to the skin surface where it evaporates, result- chamber instruments (12,19). Open-chamber instruments con-
ing in equilibrium between the ongoing process of water loss sist of a small hollow cylinder which is placed onto the skin.
at the surface and replenishment from beneath. Under physi- In still environmental conditions the water vapor from the
ological conditions the water concentration underneath and skin surface diffuses through the lower end of the chamber to
in the innermost layer of the SC is approximately 70%, and it the upper end into the ambient atmosphere. The steady-state
is approximately 10% to 30% in the outermost layer of the SC humidity gradient between the skin surface and the ambient
depending on environmental conditions (7,11,12). air is the physical basis for the water flux measurement. Using
In theory, water molecules may take two routes through these types of devices continuous measurements are possible,
the SC: diffusion via the transcellular route through the cor- but their accuracy depends on the ambient conditions.
neocytes or intercellular penetration (Figure 3.1). Because the Closed-chamber instruments have only one orifice. The
water diffusion coefficient of the intercellular lipids is consid- open end is placed onto the skin surface and the water vapor
ered to be ten times higher compared to the corneocytes, the diffuses into the closed chamber. The change of the constantly
intercellular water flux might also be higher (13). The structure increasing water concentration inside the closed-chamber is

Transcellular transport Intercellular transport Corneocytes

Skin surface


Stratum granulosum


Figure 3.1 Two-dimensional model of the human stratum corneum and possible inside-out diffusion pathways of water.

used for TEWL estimation. The accuracy of the TEWL esti- In research or clinical settings these and other details are usu-
mates is much more independent from ambient conditions, but ally specified in the standard operating procedures. These
continuous measurements are not possible. The probe needs protocols are especially important when measurements are
to be dried between measurements. The condenser-chamber conducted by many different persons, at multiple centers, and
method also uses a closed-chamber but the water vapor inside over long durations.
the chamber is controlled. Water vapor diffuses from the skin
surface toward a condenser, creating a humidity gradient
which, comparable to the open-chamber method, is used to WHAT DO TRANSEPIDERMAL WATER LOSS
TEWL estimation. Continuous measurements are possible. VALUES REALLY TELL US ABOUT THE SKIN?
Based on the different measurement principles, techni- TEWL is considered one of the most important parameters
cal specifications, measurement ranges, and degree of preci- indicating the state and integrity of the epidermal (water)
sion, different instruments produce slightly different results barrier. It is elevated in a large range of dermatological
(20). Even placing a probe onto the skin surface will influence conditions (e.g., atopic dermatitis), and TEWL decreases
the TEWL, because the water flux density on uncovered skin indicate barrier restoration and skin health (18,23). This
is different from the flux density within small measurement parameter is also very sensitive and can indicate functional
chambers (12,21). However, available method comparison changes before clinical signs become obvious (26,27). On
studies indicate that TEWL estimates seem to be comparable the other hand, there are numerous non-pathologic factors
and are highly correlated (22). influencing  the TEWL. Despite the measurement instru-
In order to achieve valid and reliable TEWL estimates, ments and measurement conditions, TEWL is dependent
measurement guidelines have been proposed (18,23–25). on many factors, including age, skin area, skin tempera-
Selected key recommendations are: ture, circadian rhythm, season, or cigarette smoking (23,25).
Because of this complexity and interdependence of the vari-
• An acclimatization period prior to TEWL measurement ous factors it is strongly recommended that TEWL values be
is required. During this time the skin areas that will be interpreted very cautiously, and not necessarily in absolute
measured must be uncovered by any material (e.g., cloth- terms (14,28).
ing). Recommended durations vary but they should not be Hundreds of descriptive and experimental studies mea-
shorter than 15 minutes. suring TEWL have been conducted, and larger scale studies
• Acclimatization and measurements should be conducted reporting skin barrier characteristics of hundreds of persons
under standardized ambient conditions. Room tempera- are now available (29,30). Two systematic reviews and meta-
tures should be lower than 22°C and the relative humidity analyses have been conducted to summarize the available evi-
should be around 50% or lower. dence about TEWL in adults of 18+ years (22) and infants from
• A calibration according to the manufacturer's instructions 0 to 24 months (31). Despite large variations, these reviews
must be conducted regularly and/or before a larger set of provide reference TEWL values for more than 50 skin areas
measurements. in healthy individuals. These reference values are useful for
• If the “baseline” or “normal” TEWL is the parameter of study planning and for comparison with existing data, but
interest, no cleansing procedures, leave-on products, or nevertheless it must be emphasized that a “normal” TEWL
similar applications must be used before measurement. seems to be unknown so far (22,23). Consequently, cutoff val-
• Neither too much nor too little pressure must be applied ues or possible TEWL thresholds are also arbitrary. According
when placing the probe head onto the skin surface. It must to Menon and Kligman “… there is no single optimal TEWL
be ensured that no gaps arise between the lower orifice value for the entire skin and that what is important is not how
and the skin. ‘tight’ a barrier is but rather when the barrier is good enough to
• The probe head should be placed vertically onto the hori- allow for survival and subserve other biological functions…”
zontal skin surface. (p. 180) (32).


A PARAMETER IN SKIN RESEARCH 1. Renbourn ET. The natural history of insensible perspiration:
In clinical research changes (delta) of TEWL over time, e.g., A forgotten doctrine of health and disease. Med Hist 1960;
during the course of disease, during artificial irritation tests, 4:135–52. Epub 1960/04/01.
or during cosmetic treatments, are much better parameters 2. Loewy A, Wechselmaa W. Zur Physiologie und Pathologie
des Wasserwechesels und der Wärmeregulation seitens des
for measuring skin barrier changes or effects of treatments
Hautorgans. Virchows Archiv F 1911; 206(1):79–121.
compared to single measurements (33,34). Whether these 3. Newburgh LH, Woodwell Johnson M. The insensible loss of
changes are large enough to be considered clinically relevant water. Physiol Rev 1942; 22(1):1–18.
is a matter of debate, but they should be larger than random 4. Pinson EA. Evaporation from human skin with sweat glands
variation (e.g., two standard deviations) in the data. While inactivated. Am J Physiol 1942; 137:492–503.
there is a clear relationship between substantially increased 5. Rothman S. Physiology and Biochemistry of the Skin. 2nd ed.
or decreased TEWL with skin barrier function and SC Chigago: The University of Chicago Press; 1955.
integrity, smaller TEWL changes might rather indicate bio- 6. Wang TF, Kasting GB, Nitsche JM. A multiphase micro-
logical variability only. For instance, a mean TEWL decrease scopic diffusion model for stratum corneum permeability. II.
of 1 g/m 2/h is unlikely to indicate “… an improvement of the Estimation of physicochemical parameters, and application to
a large permeability database. J Pharm Sci 2007; 96(11):3024–51.
skin barrier…” (p. e368) (35). Similarly, lower TEWL in aged
Epub 2007/09/197.
skin or senile xerosis should not be interpreted as a “better” 7. Igaki M, Higashi T, Hamamoto S, Kodama S, Naito S, Tokuhara
skin barrier function (p. 93) (36,37). It must be emphasized S. A study of the behavior and mechanism of thermal conduc-
that the applied TEWL measurements on the skin surface tion in the skin under moist and dry heat conditions. Skin Res
are indirect (12). Besides random variation or bias, the exact Technol 2014; 20(1):43–9. Epub 2013/06/21.
biological or functional reason for value changes must not 8. Lane ME. Emollient therapy and skin barrier function. In:
be obvious. Water diffusion through the SC is for instance Lodén M, Maibach H, eds. Treatment of Dry Skin Syndrome.
determined by the horizontal corneocyte overlap, the cor- Berlin, Heidelberg: Springer 2012; pp. 513–23.
neocyte volume and composition, or the number of layers 9. Barbero AM, Frasch HF. Transcellular route of diffusion
(10). All these parameters might not necessarily have clinical through stratum corneum: Results from finite element models.
J Pharm Sci 2006;95(10):2186–94. Epub 2006/08/03.
10. Naegel A, Heisig M, Wittum G. Computational modeling of the
In the course of cosmetic or dermatological studies, skin barrier. Method Mol Biol 2011; 763:1–32. Epub 2011/08/30.
comprehensive and transparent reporting of results is of 11. Crowther JM, Matts PJ, Kaczvinsky JR. Changes in stratum
utmost importance to ensure reproducibility, meaningful corneum thickness, water gradients and hydration by mois-
communication, and comparisons within and between stud- turizers. In: Lodén M, Maibach HI, ed. Treatment of Dry Skin
ies. Details must be reported about demographic and health Syndrome. Berlin, Heidelberg: Springer 2012; pp. 545–60.
characteristics of the sample including dermatological condi- 12. Imhof RE, De Jesus ME, Xiao P, Ciortea LI, Berg EP.
tions, season, geographical area, procedures, prior measure- Closed-chamber transepidermal water loss measurement:
ments (e.g., use of products, acclimatization), measurement Microclimate, calibration and performance. Int J Cosmetic Sci
device, the exact anatomical location, the method and num- 2009; 31(2):97–118. Epub 2009/01/30.
13. Xiao P, Imhof RE. Two dimensional finite element modelling for
ber of measurements, and ambient measurement conditions
dynamic water diffusion through stratum corneum. Int J Pharm
(23). Irrespective of the subsequent statistical analysis, TEWL 2012; 435(1):88–92. Epub 2012/02/09.
values must be reported at least using arithmetic means and 14. van der Valk PGM, Kucharekova M, Tupker RA. Transepidermal
standard deviations (22). water loss and its relation to barrier function and skin irri-
In clinical controlled intervention studies, randomiza- tation. In: Fluhr J, Elsner P, Berardesca E, Maibach HI, eds.
tion is a key design element to reduce bias. Because the TEWL Bioengineering of the Skin: Water and the Stratum Corneum. New
is strongly dependent on the anatomical location, baseline York: Informa Healthcare 2005: pp. 97–104.
comparability even between adjacent skin areas is limited. For 15. Wang TF, Kasting GB, Nitsche JM. A multiphase micro-
instance, available empirical data indicate increasing TEWL scopic diffusion model for stratum corneum permeability. I.
from proximal to distal on the volar forearm skin (22,38) Formulation, solution, and illustrative results for representative
compounds. J Pharm Sci 2006;95(3):620–48. Epub 2006/02/01.
which was supported recently by new empirical data (39).
16. Menon GK, Bommannan DB, Elias PM. High-frequency sono-
Consequently, randomization even within small skin areas is phoresis: Permeation pathways and structural basis for enhanced
complicated. Contralateral skin areas are very well compara- permeability. Skin Pharmacol 1994; 7(3):130–9. Epub 1994/01/01.
ble, and randomization should be preferred in such so-called 17. Cevc G, Vierl U. Nanotechnology and the transdermal route:
“split body designs.” A state of the art review and critical appraisal. J Control Release
2010;141(3):277–99. Epub 2009/10/24.
18. Rogiers V. EEMCO guidance for the assessment of transepider-
CONCLUSIONS mal water loss in cosmetic sciences. Skin Pharmacol Appl 2001;
TEWL is a valuable, straightforward parameter to measure 14(2):117–28. Epub 2001/04/24.
the SC integrity and the inside-out water barrier. Highly stan- 19. Sotoodian B, Maibach HI. Noninvasive test methods for epi-
dardized settings are required to yield accurate and reliable dermal barrier function. Clin Dermatol 2012; 30(3):301–10. Epub
data. This parameter is extremely sensitive to environmen-
20. Miteva M, Richter S, Elsner P, Fluhr JW. Approaches for opti-
tal factors as well as intra-individual variations among body mizing the calibration standard of Tewameter TM 300. Exp
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barrier function. Other techniques might be used in addition 21. Grove GL, Grove MJ, Zerweck C, Pierce E. Computerized evap-
to obtain a comprehensive picture of the skin barrier structure orimetry using the DermaLab TEWL probe. Skin Res Technol
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22. Kottner J, Lichterfeld A, Blume-Peytavi U. Transepidermal SC hydration, sebum content and skin surface pH. Int J Cosmetic
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Nail Penetration
Rania Elkeeb, Xiaoying Hui, and Howard I. Maibach

INTRODUCTION connects to the underlying nail bed, in which many pathological

Human nail, equivalent to claws and hooves in other mammals, changes occur. Thus, in the treatment of nail diseases, achieving
acts as a protective covering for the delicate tips of the fingers an effective drug concentration in the ventral nail plate is of great
and toes against trauma, enhances the sensation of fine touch, importance. The nail bed consists of non-cornified soft tissue
and enables one to retrieve and manipulate objects. The nail is under the nail plate. It is highly vascularized. Beneath the nail
also used for scratching and grooming, as a cosmetic organ, and, bed is the nail matrix, which is a heavily vascularized thick layer
by some, to communicate social status. The nail plate appears as of highly proliferative epithelial tissue that forms the nail plate.
a thin, hard, yet slightly elastic, translucent, convex structure (1). The human nail is approximately 100 times thicker than
Disorders of the nail resulting from conditions such as the stratum corneum, and both are rich in keratin. However,
infections or physical-chemical damage can result in painful they exhibit some physical and chemical differences (11,12). The
and debilitating states, and often change the nail plate's appear- nail possesses high sulphur content (cystine) in its hard kera-
ance. Onychomycosis, the most common nail plate disorder (2) tin domain, whereas the stratum corneum does not. The total
thickens the nail, makes it white and opaque, and in the toe- lipid content of the nail ranges from 0.1% to 1%, as opposed
nails it may cause pain while wearing shoes. Onychomycosis to approximately 10% for the stratum corneum. This suggests
is a fungal infection of the nail plate—usually caused by the that the role of the lipid pathway in the nail plate is probably of
species Epidermophyton, Microsporum and Trichophyton – and much less importance than that in the stratum corneum. The
affects 14% of the human population. Aging increases the inci- human nail acts like a hydrophilic gel membrane, while the
dence significantly, with the rate estimated at 48% in persons stratum corneum acts like a lipophilic partition membrane.
70 years of age (3). Under average conditions, the nail contains 7%–12%
To cure the infection, the patient is obliged to take oral water, in comparison to 25% in the stratum corneum. At 100%
systemic medication for an extended period, generally months, relative humidity, the maximal water content in the nail is
or undergo surgical nail removal (4). These treatments have approximately 25%, in sharp contrast to the stratum corneum
adverse effects such as pain (surgery) and systemic side effects that can increase its water content to 200%–300%. The rate of
(oral treatment). Thus, topical therapy is a desirable approach, chemical penetration into/through the human nail depends
but has met with limited success. Topical therapy is limited by upon its water solubility (11) and its molecular size (12).
the infection's deep-seated nature and by the ineffective pen- Topical therapy for onychomycosis has been largely
etration of the deep nail plate by topically applied drugs (5,6). ineffective, and this failure may be due to minimal drug pen-
How can topical drugs be delivered effectively into the etration into the nail plate (5). The nail's unique properties,
nail? And, perhaps as importantly, how can the drug content in particularly its thickness and relatively compact construction,
the human nail be assessed in order to validate nail drug delivery? make it a formidable barrier to the entry of topically applied
Our challenge was to develop a system to assay drug content in agents (6). The concentration of an applied drug across the
the inner nail bed in which infection often resides. We developed nail drops about 1000-fold from the outer to the inner surface
a micrometer-controlled drilling instrument that removes and (13). As a result, the drug concentration presumably does not
collects from the inner nail bed a powder sample from which— reach a therapeutically effective level in the inner ventral layer.
by mass balance recovery—we assay the amount of penetrated The existing clinical evidence suggests that a key to success-
radio-labeled drug. With this procedure, the effectiveness of topi- ful treatment of onychomycosis by a topical antifungal prod-
cal nail drug delivery can be assessed (7,8). This paper reviews uct lies in effectively overcoming the nail barrier. Currently
the results of studies undertaken with drilling system. available topical treatments have limited effectiveness, pos-
sibly because they cannot sufficiently penetrate the nail plate
REVIEW OF NAIL PHYSICAL AND to transport a therapeutically sufficient quantity of antifungal
CHEMICAL PROPERTIES THAT drug to the target sites (14) and eradicate the infection.
AFFECT TOPICAL PENETRATION To achieve an effective chemical concentration into/
The human nail anatomy consists of nail plate, nail bed, and through the human nail plate, penetration enhancers that tend
nail matrix. The nail plate consists of three layers: the dorsal and to promote diffusion through the skin's horny layer have been
intermediate layers derived from the matrix, and the ventral layer studied. However, these studies were conducted on a few lim-
from the nail bed (9,10). The upper (dorsal) layer, is only a few cell ited nail penetration models that may not provide an intimate
layers thick and consists of hard keratin. It constitutes a main contact between the receptor compartment and the nail sur-
barrier to drug diffusion into and through the nail plate. The face, and the nail plate can be easily hydrated beyond normal
intermediate layer constitutes three-quarters of the whole nail levels (6,11,12,14,15). Moreover, nail samples prepared with
thickness, and consists of soft keratin. Below the intermediate scalpel or sandpaper are time consuming, and may not accu-
layer is the ventral layer of soft keratin—a few cells thick—that rately represent the three nail compartment structures (10,16).

Challenges in Overcoming the technique developed by Hui et al. enables the determination of
Nail Permeation Barrier drug concentration within the plate, where fungi reside. This
Overcoming low ungual permeability to xenobiotics can method relies on a drilling system which samples the nail core
be achieved by several approaches, and is an active field of without disturbing its surface. This is achieved by the use of
research. Their effectiveness and applicability will vary from a micrometer-precision nail sampling instrument that enables
drug to drug depending on the physicochemical nature of finely controlled drilling into the nail as well as the collection
the compound. A drug absorbed topically must partition into of the powder created by the drilling process. Drilling of the
the nail bed and maintain concentration higher than the cor- nail occurs through the ventral surface. The dorsal surface and
responding minimum inhibitory concentration (MIC) to be ventrally-accessed nail core can be assayed separately. The core
effective. from the ventral side provides drug measurement at the site
A hypothesized quantitative structure–activity relation- of disease while the dorsal surface sample contains residual
ship (QSPR) model was developed based on the available in drug. This method allows for drug measurement in the inter-
vitro data set for eight drugs studied in the laboratory, listed mediate nail plate, which was previously impossible (21,22).
in Table 4.2, later in the chapter. (17) This small sample size Murthy et al. developed the TranScreen-NTM method,
weakens the predictive power of the model, and a larger sam- based on a simple and rapid technique for screening that uses
ple is needed to improve predictability. As this model has been a simple microwell plate based high throughput (23).
derived from data in one laboratory where the study design A novel model of infected nail plate made of human hair
and methodology of the nail study and nail sampling have keratin for testing the efficacy of topical antimicrobial formula-
been the same with similar sensitivity, precision, and robust- tion was recently developed by Luisiana and Muller-Goymann
ness of the analytical method using radioactivity; thus one (24). This was subsequently infected by Trichophton rubrum,
main factor contributing to a variation in ungual drug delivery the common causative agent of onychomycosis. The infected
in vitro has been eliminated. keratin films were treated with selected topical formulations—
cream, gel, and nail laquer—and compared to bovine hoof.
They demonstrated that the keratin film model was compa-
Limitations of Current Ungual Drug rable to the bovine hoof, as both gave an equivalent response
Permeability Studies (18) to the tested antifungal. The gel formulations were superior
1. Animal hooves: May not provide a representative model to the cream. Thus this model may be able to differentiate
for nail penetration in which diffusion through the human between efficacies of different topical antifungal formulations
nail can be evaluated. based on their activities against T. rubrum.
2. Nail clippings: May not provide a complete representa- Infrared (IR) and impedence microscopy (IS) tools have
tive model for nail permeation studies as they are not been studied to show their usefulness in the development and
connected to the nail bed. optimization of topical drug delivery systems in treating nail
3. Hydration-controlled method: Modified diffusion cells diseases. The authors used IR and IS to characterize the effects
of drug transport enhancement techniques such as hydration,
are a commonly used in vitro method in ungual drug
iontophoresis, and N-acetyl–L-cysteine on human nail plate.
permeation. These assays super hydrate the nail and pos-
The findings utilized nail clippings of healthy human volun-
sibly alter the physical property and nail permeability. Hui teers; further testing will be needed to characterize the spec-
et al. modified the conventional modified diffusion cells troscopic properties of diseased nail and the extent to which
commonly used for in vitro studies by using a cotton ball they are modified by the drug transport enhancement tech-
soaked in saline to provide moisture (but not saturation), nique used in the study (25).
and hydrating the nail throughout the experiment.
4. Correlation of in vitro onychopharmacokinetics assays ADVANCES IN METHODS OF IDENTIFICATION
and the in vivo onychopharmacokietics studies: The in OF THE FUNGAL ELEMENTS IN THE NAIL
vitro data obtained with the use of modified diffusion The conventional method for the identification of a fungus is
requires comparison to human in vivo data with the use of the use of KOH preparation of nail samples with consecutive
radioisotopes and possibly attenuated mass spectroscopy. culture of the nail samples on agarose plates as well as histo-
Until these correlations are defined and until we enhance logical examination of the nail plate (26). These methods are
our understanding of the pathophysiology of the disease, known to produce false negative results ranging from 20% to
biological interpretation remains tenuous (19). Additional 40%. If false negative results are obtained in a clinically evi-
references are found in Murdan 2002 and 2008 (1,20). dent onychomycosis case, an invasive nail plate punch biopsy
for giemsa staining may be performed.


Nail topical therapy has been a challenge for scientists due to Optical Coherence Tomography
the minimally permeable nature of the nail plate; thus much This method allows for noninvasive cross-sectional imaging of
interest has been directed toward finding a more efficient and the nail plate. Abuzahra et al. conducted a pilot study to com-
robust screening method. pare optical coherence tomography (OCT) with conventional
As mentioned earlier, Hui et al. modified the conventional methods. They examined the potential of OCT as a noninva-
Franz diffusion cell by using a Teflon one-chamber diffusion sive tool for the diagnosis of onychomycosis. The authors were
cell (Permegear, Inc., Hellertown, PA) along with a cotton ball able to demonstrate a reliable pattern for identifying onycho-
soaked in normal saline in order to maintain the nail at a physi- mycosis using OCT where it detected fungal elements in all
ological levels of temperature and humidity. A novel sampling histologically positive specimens, and no false positive results

were seen in their controls (27). In contrast to Abuzahra's find- to the antifungal drug, and possibly a penetration enhancer.
ings, Rothmund et al. conducted a study to evaluate the use Once the lacquer is applied, it forms a thin, water-insoluble
of confocal laser scanning microscopy (CLSM) and OCT as a film containing the supersaturated antifungal drug. This
noninvasive diagnostic tool and compare them to established provides a chemical gradient to drive drug flux as the drug
techniques. Rothmund et al. had a very low specificity and is released. Thus, a lacquer formulation is suitable for topical
a high number of false positive results with the use of OCT, treatment of nail diseases. We selected a commercial lacquer
possibly due to the lower resolution, which may not allow a formulation, EcoNail™ (MacroChem Corp). The components
clear-cut differentiation between hyphae/spores and other of this lacquer formulation include econazole with penetration
nail creases/artifacts, like trapped air, that may appear simi- enhancer, 2-n-1,3-nonyl-dioxolane (18%) and were assembled
larly (28). Contradictions are an indication for further, larger, into a test formulation in the lab prior to use (8). The control is
and more detailed investigations. the same formulation minus 2-n-1,3-nonyl-dioxolane.
[14C]-Ketoconazole was mixed with a filming solution,
Confocal Laser Scanning Microscopy Time Off Nail (Neutrogena Corporation, Los Angeles, CA) to
CLSM is regarded as a helpful noninvasive tool in the diagno- be a final 2% nail lacquer formulation. 2% [14C]-Ketoconazole
sis of skin lesions of the human skin, primarily for pigmented commercial cream was purchased from TEVA Pharmaceuticals
lesions, as well as other skin tumors and diseases. It has been USA (Sellersville, PA) and used for control.
investigated as a noninvasive tool in diagnosis of onychomy- AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxa-
cosis possibly offering higher precision and faster results com- borole) was obtained from Anacor Pharmaceuticals, Inc. (Palo
pared with KOH preparations. The study by Rothmund et al. Alto, CA). Trace amount of [14C]-AN2690 was mixed with pro-
showed that CLSM had the best specificity superior to KOH pylene glycol/ethanol (1:4, v/v) to a final 10% (w/v) test formu-
preparations, culture, and OCT (28). One major drawback to lation (30). Penlac® nail lacquer (ciclopirox 8% topical solution)
this technique is the cost. was manufactured by Dermik (Berwyn, PA) (31). Trace amount
of [14C]-ciclopirox was mixed with the lacquer to be a control.
Methodology Human Fingernail Plates
Chemicals/Formulations Nail plates were collected from adult human cadavers and
[14C]-Urea (specific activity 55 mCi/mmol, 99% purity), stored in a closed container at 0°C. Before each experiment, nail
[7-14C]-salicylic acid (specific activity 55 mCi/mmol, 99% samples were gently washed with normal saline to remove any
purity), and [3H(G)]-ketoconazole (specific activity 5 Ci/mmol, contamination, then rehydrated by placing them for 3 hours
99% purity) were purchased from American Radiolabeled on a cloth wetted with normal saline. Nail samples were ran-
Chemicals, Inc. (ARC, St. Louis, MO). [14C]-AN2690 was synthe- domly selected and allocated to test groups. Nail thickness was
sized by Amersham Biosciences UK Limited (Buckinghamshire, measured by a Sony microdigital meter (Sony Magnescale Inc.,
UK). [14C]-Econazole [chlorophenyl benzyl-14C]-(D,L)-econazole Japan) before testing in order to determine the drilling depth for
(chemical name: 1-[2-[(4-chlorophenyl)-methoxy]-2-(2,4-dichlo- each nail. Five nails were used for each formulation tested.
rophenyl)ethyl]-1H-imidazole), [14C]-ciclopirox (pyridinone-
6-(14C)-ciclopirox) was obtained from Perkin-Elmer Life Dosing and Surface Washing Procedures
Sciences, Inc. (Boston, MA). [14C]-Terbinafine freebase and A 10-µl dosing aliquot of each of the test formulations was
[14C]-terbinafine hydrogen malate were obtained from Novartis applied to the surface of a nail plate with a microsyringe.
Pharma AG (Basel, Switzerland). The radiochemical purities and Topical application was usually conducted in the morning. For
specific activities of all chemicals were >98% and >98%, respec- twice daily dosing, a second one was done in the evening, 8
tively. Penlac1 nail lacquer (ciclopirox 8% topical solution) was hours after morning application. Surface washing to remove
manufactured by Dermik (Berwyn,PA). 2-n-nonyl-1,3-Dioxolane the residue dose was done in the morning, 24 hours after the
(SEPA), a penetraton enhancer, and all necessary lacquer com- previous morning application, and 10 minutes prior to the next
ponents were provided by MacroChem Corp. (Lexington, MA). one if necessary. The nail was washed with cotton tip swabs in
a cycle as follows to simulate daily bathing: a dry swab, then
Formulations a swab wetted with 50% Ivory liquid soap (Procter & Gamble,
Nails have a high content of disulfide bonds (10.6% versus Cincinnati, Ohio), then a swab wetted with distilled water,
1.2% for human skin), which make the nails both strong and then another swab wetted with distilled water, then a final dry
impenetrable. To deliver a therapeutically sufficient quantity swab. The nails treated with lacquer also received an alcohol
of an antifungal drug to fungally-infected sites, such as nail wash to remove residual lacquer that was insoluble in soap and
plate, bed, and matrix, a suitable carrier is needed to enhance water. The samples from each cycle from each nail were pooled
drug penetration through the nail barrier. In the case of urea, and collected by breaking off the cotton swab into scintilla-
ketoconazole, and salicylic acid, a lotion (Pennsaid lotion, tion glass vials. An aliquot of 5.0 ml methanol was added to
Dimethaid Research Inc., Markham, Ontario, Canada.) con- each vial to extract the test material. The radioactivity of each
taining the penetration enhancer dimethylsulfoxide (DMSO) sample was measured in a liquid scintillation counter.
had previously been shown to enhance skin penetration
(7,8,29). To test these three drugs, we prepared three formula- Nail Incubation
tions with [14C]-urea, [3H]-ketoconazole and [14C]-salicylic acid To keep the nail at physiological levels of temperature and
at 0.002%, 0.1%, and 0.07%, respectively, and corresponding humidity, we incubated it in a Teflon one-chamber diffusion
saline controls with each drug at the same concentrations (7). cell (PermeGear, Inc., Hellertown, PA). The nail surface (top
For the antifungal drugs econazole, terbinafine free base, center) was open to air and the inner surface made contact with
and terbinafine salt, we used a nail lacquer formulation, which a small cotton ball acting as a nail supporting bed (Figure 4.1).
is a popular choice for topical antifungal treatment. Nail lac- The cotton ball was wetted with normal saline. The incubation
quer contains a film-forming agent and a solvent, in addition period started 24 hours prior to the first dose, and ended 24

hours after the final dose. A small cotton ball wetted with 0.1 After completion of the dosing and incubation phase,
mL normal saline was placed in the chamber beneath the nail the nail plate was transferred from the diffusion cell to a clean
plate to serve as a “nail bed” and provide moisture for the nail cutting holder for sampling. The nail plate was secured in
plate, and hydration was monitored and controlled during the position so that the ventral surface faced the cutter and the
experiment (7,8). This cotton ball method prevented overhy- dorsal-dosed surface faced the holder. The cutting holder was
dration of a liquid interface. Cotton is a hydrophilic fiber which moved to bring the plate surface just barely in contact with the
can contain a high degree of moisture content and absorbs cutter tip. The drill was then turned on and a fine adjustment
water-soluble substances. It also absorbs lipophilic substances, moved the stage toward the cutter tip, removing a powder
which can fill the lumen and lie between the numerous inter- sample from the nail. In this way, a hole approximately 0.3–0.4
nal layers of the cotton (32). Thus the cotton ball is an ideal mm in depth and 7.9 mm in diameter was drilled in each nail,
receiving medium for in vitro transungual delivery. enabling the harvest of powder sample from the center of each
nail's ventral surface. We refer to these samples as having been
Nail Sampling
taken from the “ventral/intermediate nail plate.”
The objective was to determine drug concentration within
After the nail had delivered its ventral/intermediate
the nail where the disease resides. Treatment is applied to the
nail plate powder samples, it was removed from the sampling
nail surface. The drilling system samples the inner core of the
instrument. The nail outside the dosing area was cut away
nail without disturbing the nail surface. The two parts (sur-
and discarded. The nail within the dosing area but outside
face and inner core) can be assayed separately. The surface
the sampling area was trimmed away and saved; we refer
contains only residual drug after washing. The drilled-out
to this as the “remainder nail plate.” It surrounds the dorsal
core (from the ventral side) is thus a true drug measurement
layer above the sampling area where the powder samples were
at the target site where the disease resides (Figure 4.2). Drug
taken; we refer to this as the “sampling area dorsal nail plate.”
penetration into the nail was sampled by a unique microm-
eter-controlled nail sampling instrument that enabled finely
controlled drilling into the nail and collection of the powder
created by the drilling process (7,8). The nail sampling instru- Dorsal layer Topical dose Dorsal/intermediate center
ment (Figure 4.3) has two parts, a nail sample stage and a drill.
Intermediate layer
The nail sampling stage consists of a copper nail holder, three
adjustments, and a nail powder capture. The three adjust- Remainder nail
ments control vertical movement. The first coarse adjustment
(on the top) is for changing the copper cell and taking powder
samples from the capture. The other two adjustments (lower)
are used in sampling. The second coarse adjustment allows Ventral layer Ventral/intermediate center
movement of 25 mm while the fine adjustment provides Cutting tip
movement of 0.20 mm. The nail powder capture is located for nail sampling
between the copper cell and the cutter. The inner shape of the
capture is an inverted funnel with the end connected to a vac-
Figure 4.2 Nail and nail drilling tip.
uum pump. By placing a filter paper inside the funnel, nail
powder samples can be captured on the filter paper during
sampling. The nail is fastened in a cutting holder below the
cutter and surrounded by a funnel containing a filter paper.
The funnel is attached to a vacuum pump. During drilling,
the vacuum draws the powder debris onto the filter paper so
it can be collected and measured and increase total collection
for mass balance determination.

Cotton ball Top cover & metal holder Nail chamber

Figure 4.1 Nail support and incubation system in a Teflon one-

chamber diffusion cell. The cotton ball prevented the over hydra-
tion of a liquid interface. Cotton is a hydrophilic fiber which can Figure 4.3 The nail sampling system. The instrument has two
contain a high degree of moisture content and absorbs water- parts, a stage and a drill. The stage consists of a copper nail
soluble substances. It also absorbs lipophilic substances, which holder, three adjustments, and a nail powder capture. During
can fill the lumen and lie between the numerous internal layers of drilling, the vacuum draws the powder debris onto the filter paper
the cotton (32). Thus the cotton ball is an ideal receiving medium so it can be collected and measured and increase total collection
for in vitro transungual delivery. for mass balance determination.

The ventral/intermediate nail plate powdered samples, the which is an effect of the drug delivery enhancer. At the sam-
sampling area dorsal nail plate, and the remainder nail plate pling area dorsal nail plate, there is more econazole from the
were individually collected into a glass scintillation vial and saline control because the dose remained on the nail surface.
weighed. The nail samples were then dissolved by adding 5.0
mL of Packard's Soluene®-350 (Packard Instrument Company, Effects of Dosing and Washing Frequency
Meriden, CT). The total mass of nail collected was measured Figures 4.4 and 4.5 show the weight normalized of 14C econ-
by the difference in weight of the plate before and after drill- azole equivalent in different layers of the nail plate and cumu-
ing (7,8). lative 14C econazole equivalent collected in the cotton ball
supporting bed following different frequency of topical dosing
Results or surface washing treatments. As expected, after twice daily
Nail Incubation Conditions application (and surface washing once daily) for 14 days, the
14C econazole content in all nail and cotton ball samples was
Table 4.1 shows that the average hydration of the wetted cot-
ton balls 109 ± 6.2 AU (arbitrary units [a digital expression of significantly higher than that in the dosing and washing once-
capacitance]) that resembles the average hydration of human daily group (p < 0.05). Comparison of the once-daily dosing
nail bed, 99.9 ± 8.9 AU measured from fresh human cadav- and washing treatment group and the once-daily dosing and
ers. During the experiment, the holding tank temperature once-weekly washing group did not show significant differ-
was 25 ± 2ºC and relative humidity was 44 ± 8%. Thus, there ence for all collected samples (p > 0.05).
was no statistical difference between hydration conditions
Enhancer Effects
for nails treated with either the test formulation or the saline
Table 4.4 summarizes the penetration of ketoconazole, urea,
control. This incubation device is nonocclusive and hydra-
and salicylic acid into the human inner nail plate. Each test
tion controlled, and approximate normal physical condition
formulation contained a drug delivery enhancer (7,8) and was
is reached.
compared to a control formulation without any penetration
Accuracy of Nail Sampling Process enhancer. In each case the test formulation enhanced drug
The advantage of the micrometer-controlled drilling and delivery (p < 0.05). Table 4.5 compares antifungal efficacy of
nail powder removal system is the accuracy of the sampling econazole, terbinafine base, and terbinafine salt following
process. The sampling instrument allowed well-controlled, topical applications. Each test antifungal agent contains 18%
accurate, and reproducible sampling of the inside of the nail. 2-n-nonyl-1,3-dioxolane (SEPA), a skin penetration enhancer,
Table 4.2 shows that the average depth of nail sampling from and the control (no 18% 2-n-nonyl-1,3-dioxolane). All test for-
the inner center surface was well controlled at 0.26 ± 0.05 mm, mulations show the antifungal efficacy coefficient (E) was sig-
which was close to the expected depth of 0.24 mm. The weight nificantly higher than that of corresponding control (p < 0.05).
of the nail samples collected was consistent for all experiments.
Topical Formulation Comparison
Mass Balance of Radioactivity Recovery Table 4.6 compares [14C]-ciclopirox penetration into/through
Table 4.3 summarizes the econazole mass balance recovery fol- nail plate in vitro from three topical formulations: market gel,
lowing the 14-day nail treatment. Overall recovery of applied experimental gel, and lacquer. The order of deeper penetra-
dose was 90.8 ± 16.4% for the test formulation and 96.4 ± 7.3% tion, amount detected in the ventral/intermediate nail center,
for the saline control, indicating that essentially the entire dose and cotton ball supporting bed samples was from the highest
was accounted for. to the lowestmarket gel, lacquer, and experimental gel, respec-
Table 4.3 also indicates what happens to chemicals tively. Figure 4.6, however, shows that the deeper penetration
applied to the nail. Approximately 72% was washed from the of a lacquer formulation of ketoconazole was greater than that
surface. The dose absorbed from the surface of the nail pene- of cream when comparing their antifungal efficacy coefficient
trated to the sampling area dorsal nail plate (11.4%), the ventral/ (p < 0.05).
intermediate nail plate (1.4%), and the supporting bed (0.7%),
which is the cotton ball upon which the nail rested. Note that DISCUSSION
econazole recovery in the test formulation is greater for both Topical therapy for onychomycosis is not yet maximally effective,
the ventral/intermediate nail plate and the supporting bed, and this failure may be due to inadequate penetration of drugs

Table 4.1 Hydration of Nail Plate and Nail Bed

Measurement* Hydration (AU)**

Source N Time Nail plate Nail bed
Human cadavers 6 24-hr post mortem 7.6 ± 0.9 99.9 ± 8.9
Diffusion cells 8 Twice/day for 7 days 8.5 ± 2.4 109.9 ± 6.2

Note: During the experiment, the holding tank temperature was 25 ± 2°C and relative humidity was 44 ± 8%. The importance of this controlled tem-
perature and humidity is to mimic normal physiological condition of the human nail to prevent over hydration which was easily occurred in nail in vitro
*Hydration of the nail plate and the supporting cotton bed was measured with a Corneometer CM 820 (Courage & Khazaka, Cologne, Germany).

**AU, arbitrary units—a digital expression of capacitance. Thus the Corneometer CM 820 gives only an estimate of the nail (or other membrane such

as stratum corneum) hydration. Agache et al. (33) used a sorption–desorption test to assess the water content of the stratum corneum. They corre-
lated the results measured with the Corneometer CM 820 (as AU unit) and TEWL (g.m–2.h–1) measured with an evaporimeter and found that water
retained in the SC(µ–2) = In(AC/3.8)/0.0436.

Table 4.2 Nail Core Sampled from the Ventral (Inner) Surface Center of the Human Nail Plate

Nail core sampled from the ventral (inner) surface center of the nail plate*
Whole nail Depth of core % Whole nail Total core sample Powder sample
Test thickness (mm) (mm) thickness removed (mg) collected (mg)
Urea (control) 0.65 0.25 39.52 16.4 5.2
(0.09) (0.03) (8.05) (4.3) (0.8)
Urea (test) 0.71 0.27 37.97 17.6 6.4
(0.07) (0.03) (2.69) (4.3) (1.3)
Ketoconazole (control) 0.68 0.28 41.88 14.3 6.7
(0.05) (0.03) (1.16) (6.7) (2.6)
Ketoconazole (test) 0.73 0.28 38.62 14.1 4.3
(0.03) (0.02) (2.69) (5.1) (1.6)
Salicylic acid (control) 0.77 0.25 32.62 12.1 6.0
(0.07) (0.08) (9.38) (2.4) (0.5)
Salicylic acid (test) 0.60 0.21 35.03 23.4 4.7
(0.12) (0.06) (6.45) (8.3) (0.8)
Average 0.69 0.26 37.61 16.3 5.5
(0.09) (0.05) (6.20) (6.2) (1.6)

Source: Hui X et al., J Pharm Sci; 91:189–95, 2002.

*Nail sample, approximately 0.24 mm in depth and 7.9 mm in diameter, was drilled from the center of the ventral surface of the nail. The amount of

nail sample removed was measured by difference in weight and depth of the drilled area before and after sampling. Each number represents mean
(± S.D.) of 5 samples. The data demonstrated the repeatability and accuracy of the nail sampling system.

Table 4.3 Mass Balance Recovery of Econazole following 14-Day Human Nail Treatment with a Test Formulation Containing a
Penetration Enhancer and a Control without a Penetration Enhancer

Carbon-14 recoveryas percent of dose

Sampling Area Test Formulation Control Formulation
Dorsal//intermediate nail plate 11.4 (3.6) 20.1 (2.9)
Ventral/intermediate nail plate (powdered 1.3 (1.1) 0.22 (2.9)
Remainder nail plate 5.6 (3.9) 3.2 (2.3)
Supporting bed (cotton ball) 0.7 (0.3) 0.0 (0.0)
Surface washes 71.7 (12.5) 72.8 (5.1)
Total 90.8 (16.4) 96.4 (7.3)

Source: Hui X et al., J Pharm Sci; 92:142–8, 2003.

Note: The data represent the mean (SD) of each group (n = 5). The test formulation group contains 18% 2-n-nonyl-1,3-dioxolane and the control
formulation contains no 2-n-nonyl-1,3-dioxolane. The data demonstrated not only the importance of the penetration enhancer but the high recovery
rate of two groups when using the nail sampling system.

into the nail plate. The nail's unique properties, particularly its example, 14C-ketoconazole lacquer yields a significantly higher
thickness and relatively compact construction, make it a formi- antifungal efficacy coefficient than that in cream (p < 0.05;
dable barrier to the entry of topically applied agents. The concen- Figure 4.6). However, the deeper penetration of [14C]-ciclopirox
tration of an applied drug across the nail drops about 1000-fold from the commercially available formulation, Penlac nail lac-
from the outer surface to the inner surface. As a result, the drug quer (ciclopirox 8% topical solution), was statistically lower
concentration presumably does not reach a therapeutically effec- than that of market gel formulation (ciclopirox 0.77 % topical
tive level in the ventral/intermediate layers. To optimize the nail solution) (Table 4.6) (31). When compared with other antifun-
penetration of topical treatments, it is important to consider the gal topical formulations, such as [14C]-AN2690, 10% (w/v) in
nail's unique barrier properties and develop an antifungal drug propylene glycol/ethanol solution, it decreased (p < 0.05) the
formulation that has matching physicochemical properties. deeper nail layer penetration rate (Figure 4.7) (30).
Nail lacquer formulations, a popular choice for topical Transungual enhancement has been examined: DMSO
antifungal treatment, typically contain a film-forming agent, and 2-n-nonyl-1,3-dioxolane used as transdermal deliv-
solvent, antifungal drug, and possibly a penetration enhancer. ery enhancers were tested for enhancement of antifungal
Once the lacquer is applied, it forms a thin film containing a agents penetrating into and through the nail plate in vitro.
supersaturated antifungal drug. This film provides a chemi- The two enhancers increased the deeper nail penetration of
cal gradient to drive drug flux as the drug is released. For [14C]-ketoconazole (Table 4.3), [14C]-econazole, [14C]-terbinafine

* Dorsal/intermediate nail center
Ventral/intermediate nail center

Weight normalized 14C-econazole

Cotton ball supporting bed

equivalent (µg/g nail)



Twice daily Once daily
Dose frequency

Figure 4.4 Nail penetration profile of 14C-econzaole following a 14-day treatment/incubation period in vitro. Each bar represents the
mean (SD) of 6 samples. The frequency of topical dosing was different but the surface wash was the same, once daily. *The group that
received twice-daily topical doses was statistically significantly higher than the one treated with a once-daily dose (p < 0.05).

(36). However, [14C]-dioxolane, the radiolabeled 2-n-nonyl-

1,3-dioxolane, had minimal penetration to and through the
human nail plate in vitro (8). 2-n-nonyl-1,3-dioxolane can func-
Weight normalized 14 C-econazole equivalent

Dorsal/intermediate nail center

60000 Ventral/inermediate nail center tion as an adhesion promoter and a plasticizer for the film-
Cotton ball supporting ball forming polymer of the nail lacquer (37,38). The enhancement
50000 function of 2-n-nonyl-1,3-dioxolane for the tested antifungal
agents was possibly to soften the lacquer film to increase releas-
ing per-unit time (8). As shown in Table 4.5, the amounts of
(µg/g nail)

[14C]-econazole, [14C]-terbinafine free base, and [14C]-terbinafine
30000 salt detected from the deeper layer, ventral/intermediate nail
layer in the test groups, which the lacquer formulation con-
20000 tains 18% 2-n-nonyl-1,3-dioxolane were significantly greater
than that in the controls (p < 0.05). The results suggest that the
10000 enhanced level of these antifungal agents in the ventral/inter-
mediate layers and supporting bed dramatically increased,
0 which exceeds the minimum inhibitory concentration (MIC)
Once daily Once weekly
of econazole for most common onychomycosis organisms
Surface washing frequency
(Table 4.5).
MIC is a laboratory index in the determination of anti-
Figure 4.5 Nail penetration profile of 14C-econzaole following a fungal potency. Mertin and Lippold (11) introduced an effi-
14-day treatment/incubation period in vitro. Each bar represents cacy coefficient E to better estimate and compare the relative
the mean (SD) of 6 samples/group. Each group received once- efficacy of antifungal agents. The efficacy coefficient E is the
daily topical application for 14 days. The frequency of surface
ratio of the flux of an antimycotic drug through the nail plate
washing for each group was different. The group that received
to the MIC. For econazole, the range of MIC for the dermato-
daily washing was not statistically significant from the correspond-
ing bar from the once-weekly washing group (p > 0.05). phyte species is 0.1 to 1.0 µg/mL and for yeast species it is 1.0
to 100 µg/mL (39). After 14 days of exposure, the econazole
content measured in the test group was 11.15 ± 2.56 µg/mg
for the ventral/intermediate layers. This content, multiplied
by the density of the nail sample (1.332 mg/cm3, measured
salt, [14C]-terbinafine base (Table 4.5) significantly compared to under current experimental conditions), yields 14,830 ± 340
the controls (p < 0.05). DMSO has been previously reported to µg/cm3 of econazole, almost 15,000 times the MIC for most
facilitate the penetration of some topical antimycotics (15,35). dermatophyte species and 150 times that for most yeast spe-
The enhancement function of 2-n-nonyl-1,3-dioxolane has not cies (Table 4.5).
previously been determined. The mechanism of 2-n-nonyl- This study demonstrated that with our in vitro nail study
1,3-dioxolane in skin penetration was suggested to reversibly methodology, the nail plate can be scientifically studied, and
fluidize the stratum corneum lipids and alter barrier function with proper formulation one can deliver a variety of chemicals,

Table 4.4 Radiolabeled Drug Penetration into Human Nail from a Test Formulation Containing DMSO, a Penetration Enhancer, versus
a Control without a Penetration Enhancer

Radioactivity content in ventral/intermediate center layer of the nail plate

Penetration Control Significant
Test chemicals Enhancer* Unit** Test formulaton formulation (p < 0.05)
Ketoconazole Dimethylsulfoxide µg 53.9 34.0 Yes
Eq/g (10.6) (15.9)
Urea Dimethylsulfoxide µg 0.3 0.2 Yes
Eq/g (0.1) (0.1)
Salicylic acid Dimethylsulfoxide µg Eq/g 10.2 7.0 Yes
(0.6) (1.1)

Source: Hui X et al., J Pharm Sci 91:189–95, 2002.

Note: The data represent the mean (SD) of 5 samples per formulation group. The nail sample drilled as powder from ventral/intermediate layer of
human nail plate.
*Dimethylsulfoxide (DMSO) is a transdermal delivery enhancer. The enhancement mechanism of transungual delivery is not clear. However, it did

enhance the nail penetration of the test chemicals.

**µg Eq/g = microgram equivalents drug per gram of nail sample. Because radioactivity is used, the drug mass is referred to as “equivalents” because

radioactivity was measured, not the drug itself.

Table 4.5 Comparison of Econazole Concentration and Relative Antifungal Efficacy with a Test Formulation Containing 2-N-Nonyl-
1,3-Dioxolane, a Penetration Enhancer, and a Control

Parameter* Test formulation Control formulation Significant(p < 0.05)

Econazole in the deeper layer 14,830 (341) 2,371 (426) Yes
ED (MICD = 1 µg/mL) 14,830 2,371 Yes
EY (MICY = 100 µg/mL) 148 23.7 Yes
Terbinafine salt in the deeper 5946 (1029) 463 (271) Yes
layer (µg/cm3)
ED (MICD = 0.04 µg/mL) ** 5946 463 Yes
EY (MICY = 1.77 µg/mL) ** 134 10 Yes
Terbinafine base in the deeper 727 (372) 407 (106) Yes
layer (µg/cm3)
ED (MICD = 0.04 µg/mL) c 1527 156 Yes
EY (MICY = 1.77 µg/mL) c 34 3 Yes

Note: The data represent the mean (SD) of each group (n = 5). The test formulation group contains 18% 2-n-nonyl-1,3-dioxolane and the control
formulation contains no 2-n-nonyl-1,3-dioxolane.
Abbreviations: E, antifungal efficacy coefficient; MIC, minimum inhibitory concentration (of the tested antifungal agent); D, dermatophytes; Y, yeast.
(Reference 8)
*The deeper layer is the center of the ventral/intermediate layer of the nail plate. The data represent the amount of drug in the sample after a 14-day

dosing period. The amount of antifungal agents in the tested nail layer (µg/cm3) was computed from the average of the cumulative amount of the test
agent permeated into the area (area × thickness) of the deeper layer of the nail (dorsal/intermediate layer).
**See Reference 34.

Table 4.6 Summary of Weight Normalized 14C-Ciclopirox Equivalent in the Nail and Supporting Bed Samples after 14-Day Treatment

Normalized 14C-Ciclopirox Equivalent

Items (Unit) Marketed Gel Experimental Gel Lacquer Significant (if P value < 0.05)
Dorsal/intermediate center within 71.7 103.4 2162.1 Lacquer vs experimental gel
surface of nail (16.8) (38.1) (526.4) Lacquer vs marketed gel
(μg eq/mg)
Marketed gel vs lacquer
Ventral/intermediate center within 0.6 0.2 0.3 Marketed gel vs experimental gel
infection-prone area (0.3) (0.1) (0.1) Experimental gel vs lacquer
(μg eq/mg)
Marketed gel vs lacquer
Penetration through the nail into the 46.3 6.0 15.5 Marketed gel vs Eexperimental
supporting bed cotton ball (4.1) (1.3) (3.1) gel
(μg eq/sample) Experimental gel vs lacquer

Source: Hui X et al., J Pharmaceut. Sci; 93:2545–8, 2004.

Note: The data represents the mean (SD) of each group (n = 5).

Taken together, the methodology appears robust; as

500 with other models, inclusion of other chemicals with varying
physicochemical properties, and in vivo replication will add
ED (MIC = 0.125 µg/mL) strength to the results.
Efficacy coefficient (E) of ketoconazole

* EV (MIC = 0.190 µg/mL)


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Section II
Pharmacology of Cosmetic Products and Ingredients

Sensitive Skin: New Findings Yield New Insights

Miranda A. Farage and Howard I. Maibach

INTRODUCTION to tests of objective irritation (10). The diversity of methodology

The goal of product testing is to ensure that consumer products approaches employed in sensitive-skin research has also
are free of irritant potential and to prevent unexpected contributed to interpretive difficulties. Further, much of the
consumer reactions to the product once it reaches the market- research to date has been published in cosmetic journals that
place. It is not uncommon, nonetheless, for postmarketing sur- are inaccessible through major databases and may be ignored
veillance efforts to receive reports of sensory perceptions not by leading dermatological publications (5).
predicted by even the most robust methodology (1). These sen- Irritant testing reveals profound interpersonal vari-
sory perceptions, though often transient and not accompanied ability in individual response to specific irritants (11,12), even
by a visual dermatological response in rigorous experimental among chemicals with similar modes of action (13). Some have
testing, strongly influence consumer product preference (1). In reported sizeable variation within the same individual at dif-
fact, 78% of consumers who profess sensitive skin report avoid- ferent anatomic sites (12), and even at the same anatomical site
ing some products because of unpleasant sensory effects asso- on symmetric limbs (14).
ciated with their use (2). The etiology of sensitive skin is unknown, but the dis-
Unpleasant and subjective sensory effects, often unac- order is believed to be the product of multiple etiologies with
companied by objective physical signs, define a controversial multiple components, including deficiencies in barrier func-
and still evolving dermatological condition known as sensi- tion, neurosensory dysfunction, compound-specific irritancy,
tive skin. Consumer reports of sensitive skin are typically self- and cultural influences (15).
diagnosed and may be increasing (3). The concept of sensitive Deficiencies in barrier function have been shown to be
skin arose in the 1970s with the observation that despite the a critical component of skin discomfort (16). Increased perme-
fact that previous safety evaluations had found no evidence ability is believed to be the result of a functional compromise
of toxicity, some patients reported stinging sensations upon of barrier function in the sensitive-skin patient (17). Lipid
using a particular sunscreen that contained a derivative of content of the stratum corneum has been shown to be a more
P-aminobenzoic acid (4). Growing consumer awareness of the accurate predictor of skin permeability than stratum corneum
potential for irritation from common products fueled a huge thickness or cell number (12). The permeability barrier in the
increase in products marketed for the supposedly rare indi- stratum corneum requires the presence of well-organized
vidual with susceptibility to components of everyday prod- intracellular lipids (7,16) and depends highly on lipid composi-
ucts, particularly since no requirement for proof of safety and tion (12). Increased neutral lipids and decreased sphingolipids
efficacy for such products has existed (5). The initial conceptu- are associated with superior barrier properties (12). A weak
alization of sensitive skin as a minority complaint has not been barrier inadequately protects nerve endings and facilitates
borne out by epidemiological surveys, which consistently find access to antigen presenting cells, a mechanism which would
a high prevalence of sensitive skin across the world (Table 5.1). support an association with atopic conditions (16). Irritation
Sensitive skin is a term whose definition continues to be results from the abnormal penetration in skin of potentially
refined. It is now generally agreed to describe unpleasant sub- irritating substances and a resulting decrease in the skin toler-
jective sensory reactions (such as, prickling, burning, tingling, ance threshold (7). Alterations in barrier function in sensitive-
or pain) in response to common external factors (such as ultra- skin patients have been observed (18,19). Alterations of baseline
violet light, heat, cold, wind, cosmetics, cleaning products, etc.) capacitance values imply barrier impairment and support the
and intrinsic stressors (such as stress or hormones) (6). Sensory view that hyperreactivity to water-soluble irritants results
effects are only occasionally accompanied by erythema or from increased absorption (20). A derangement of intercellular
other demonstrable irritation or immunological responses (6). lipids, specifically, was also associated with a decline in bar-
Although sensitive skin is now generally accepted as a rier function in sensitive skin (21). The pain sensations, which
real physiological disorder, there is still no consensus regard- are the hallmark of the disorder, also imply possible integra-
ing its etiology, classification, or criteria for diagnosis. Several tion dysfunctions in the central nervous system. Cho et al. (22)
issues have hindered a better understanding of this condition. found that individuals were classified into sensitive skin and
It is typically self-diagnosed (7). Patients may interpret an non-sensitive skin based on a lactic acid sting test performed
underlying dermatological condition as well as any reaction on the face. There were no differences in transepidermal water
to product use as sensitive skin (8). There are also some psy- loss (TEWL) and erythema index values between the two
chological disorders characterized by similar symptoms (e.g., groups. However, the mean value of the quantity of stratum
cosmetic intolerance syndrome, dermatological non-disease) corneum ceramides on the face was significantly lower in the
(9). Many people who profess sensitive skin do not predictably sensitive skin group compared to the non-sensitive group.
experience visible signs of the sensations reported, whereas The pain and other unpleasant sensations that are
some who describe themselves as non-sensitive react strongly the hallmark of sensitive skin imply a likely relationship to

Table 5.1 Prevalence of Sensitive-Skin Perception in Various Geographies

Percentage of people who

Population(s) studied Population characteristics Definition of sensitive skin claimed sensitive skin Reference(s)
France (2000) 319 women, conducted by Cutaneous discomfort in 90% (23% very sensitive skin) 4
interviews the absence of clinical
and histological evidence
of skin lesions
France (2005) 1006 men and women, Sensitive skin 59% women, 44% men 36
identified by questionnaire
France (2006) 8522 men and women, Sensitive skin 61% women, 32% men 67
identified by questionnaire
France (2008) 1004 men and women, Sensitive scalp 44.2% (47.4% women, 40.8% 70
opinion poll survey men)
France (2008) 18 women, identified by Sensitive skin 50% (facial skin, specifically) 65
France (2008) 400 identified by Sensitive skin 85% (facial skin, specifically) 6
Germany (2001) 420 men and women Sensitive skin 75% (48% severe) 60
England (2001) 3300 men and women, Intolerance to cosmetics 51.4% men (5.8% very 32
conducted by mailed and toiletries, including sensitive skin)
questionnaire both sensory and visible 38.2% women (10% very
signs sensitive skin)
Italy (2005) 1870 women Sensitive skin 56.50% 31
Greece (2008) 25 women, identified by Sensitive skin 64% 61
Greece (2008) 25 women with atopic Sensitive skin 100% 61
USA (San Francisco) 800 women, conducted by Sensitive facial skin 52% 2
(2002) telephone interview
USA (Cincinnati) 1039 men and women Sensitive skin 68.4% reported some degree 3
(2009) surveyed by of sensitivity (“slight”–“very”)
questionnaire (69.0% women, 64.4% men)
USA (Cincinnati) 29 women with light Sensitive skin 83% reported some degree of 71
(2006) incontinence sensitivity (“slight”–“very”)
USA (2011) 994 subjects by phone Rate their skin as: “very 44.6% reported “very 70
survey (495 men and 499 sensitive,” “sensitive,” sensitive” or “sensitive”
women) “slightly sensitive,” or “not (38.2% men, 50.9% women)
sensitive at all”
USA (2013) primarily 89 women by questionnaire Sensitive skin 77.5% reported some degree 72
rural southern states of sensitivity (“slight”–“very”)
Japan, USA, Europe 15 000 men and women, People whose skin reacts to 50% women (25% very 73
(1992) conducted by particular insults more sensitive skin) 30% men
questionnaire than the majority of
China (2012) 408 women surveyed by Sensitive skin 23% 74
China (urban dwellers) 9154 surveyed by Sensitive skin 13% (8.62% men, 15.93% 75
(2012)* questionnaire (3931 men women)
and 5223 women)
Mexico (2013) 246 subjects (78 man and Sensitive skin 36% 76
168 women)
Dermatologist opinion
USA (2013) 300 dermatologists Sensitive facial skin 58.3% have noticed an 77
surveyed by email increase in male patients
questionnaire reporting sensitive facial
skin over the past 5 years
Europe (2013) 1531 dermatologists Sensitive facial skin 82% have noticed an increase 77
surveyed by in male patients reporting
questionnaire sensitive facial skin over the
past 5 years

*Year of publication, not year of study.


dysfunctions in the central nervous system, possibly involving sensitivity, as thickness of the epidermis was observed to be
several neuromediators and sensory receptors (23). Of the two greater in males than in females (p < 0.0001) (27), and hor-
studies reviewed that did evaluate the relationship between monal differences, which may produce increased inflam-
neurosensory responses and objective clinical irritation and matory sensitivity in females, have also been demonstrated
included only subjects with demonstrated sensory sensitivity, (14,28). Irritant testing, however, generally finds no differ-
both showed a correlation between sensory and objective signs ences (12). One study, though, found among 1039 subjects
(18,19). In a study regarding sensitivity to facial tissue which a 68.4% prevalence of self-reported sensitive skin, with no
did not exclude non-sensitive individuals, sensory effects were difference between men and women (3). It may be that with
demonstrated be the most reliable measure of product differ- increased marketing of products for sensitive skin in men
ences (18,19). it has become more cultural acceptable for males to define
Although no predictive value was demonstrated for themselves as having sensitive skin.
any individual sensitivity when subjects were tested with a
seven-irritant panel, a weak association between tests was
demonstrated by statistical analysis of binomial probability
The physiological changes that occur as skin ages would
(13). However, studies which evaluated the association of
predict an increased susceptibility to irritants (29). Existing
barrier function and sensitivity have yielded arguably the
studies, however, are ambiguous with regard to the influ-
most conclusive results. A high baseline TEWL was associ-
ence of age on skin sensitivity. Clinical assessment of the
ated with increased susceptibility to numerous cutaneous
erythematous response to irritants in older people suggest
irritants by numerous studies and a variety of assessment
that susceptibility generally decreases with age (29). However,
methods (14).
objective signs of irritation often show little correlation with
Most methods focused on objective assessment of physi-
the intensity of subjective complaints (29). A study of sensory
cal effects to skin rather than the sensory effects reported
perceptions of sensitive skin conducted on 1029 individu-
(24), and few reports have quantified sensory effects or cor-
als in Ohio stratified subjects into four age groups (subjects
relate sensory effects to degree of irritation. Most testing has
under 30, in their 30s, in their 40s, and over 50), and evaluated
included few subjects, and few have restricted subjects to
subjective data according to age (29). Those over 50 were more
those with demonstrated sensitivity (25). Few have attempted
likely to claim sensitive skin than younger adults and more
to evaluate the influence of endogenous hormones or lifestyle
likely to perceive genital skin (to the exclusion of other body
sites) to be more sensitive (29). Older adults also stated that
Ultimately, traditional irritant-testing methodologies
their skin had become more sensitive over time (46%) (29). The
have not proven to be good predictors of consumer response
turnover rate of the stratum corneum has been reported as
(1). Response to one irritant does not predict sensitivity to
longer in aged skin (30), which may contribute to increased
another, and has not correlated well with evaluation of objec-
sensitivity. However, in a large Italian study that performed
tive signs (26).
lactic acid sting tests on more than 100 elderly subjects, the
intensity of the stinging response was inversely proportional
Numerous potential host factors (Table 5.2) undoubtedly play
a role in experimental variability observed in sensitive skin. To
date, no constitutional factors have been identified (23). Ethnicity
There are pronounced differences in skin structure depend-
Gender ing on skin type (Table 5.3), and racial differences, with regard
Sensitive skin is self-reported far more often in women than to skin susceptibility to irritants, are among the fundamental
in men (Table 5.1). There is biological plausibility for greater questions in dermatotoxicology (25). Two large epidemiologi-
cal studies reported no observed racial differences in reporting
Table 5.2 Possible Contributors to Sensitive Skin product sensitivity (2,32). Most testing, however, has focused
Factor Reference(s) on Caucasian females (25).
Differences have been observed in sensory percep-
Female sex 32 tions, although substantive conclusions are hard to provide.
Hormonal status 44
Asians have been reported to complain of unpleasant sensory
Cultural expectations in technologically 60
advanced countries responses more often than Caucasians, supported by the obser-
Fair skin which is susceptible to sunburn 34 vation that a higher incidence of dropouts in a Japanese clinical
Susceptibility to blushing and/or flushing 32 study withdrew because of adverse skin effects as compared
Skin pigmentation 34 with those in Caucasian studies (33). There have also been
Thin stratum corneum 25, 33, 78, 79 reports of an increased sensory response, as well as speed of
Decreased hydration of stratum corneum 7, 80, 81 response in Asian subjects versus Caucasian in sensory test-
Disruption of stratum corneum 34 ing (33). Another study, however, found fair-skinned subjects
Increased epidermal innervations 7, 63 prone to sunburn had higher sensory responses to chemical
Increased sweat glands 33 probes than those with darker skin tones (34). No racial differ-
Increase neutral lipids and decreased 82
ences in innervation on an architectural or biochemical level
Decreased lipids 20, 83–86 have been observed (13).
High-baseline TEWL 14 Studies of racial differences with regard to irritants
Atopy 59, 61 have yielded conflicting evidence. Although black skin was
demonstrated to have greater potential for irritant susceptibil-
Abbreviation: TEWL, transepidermal water loss ity than white skin (12), another study found blacks to be less

Table 5.3 Comparison of Racial Differences in Functional Skin Properties

Skin property Types Racial differences Reference(s)
Permeability In vitro penetration of fluocinolone Lower in blacks than in Caucasians 87
In vitro penetration of water No differences 87, 88
Topical application of anesthetic mixture Less efficacy in blacks than in Caucasians 89
In vivo penetration of C-labeled Lower in blacks (34%) than in Caucasians 90
Methyl nicotinate-induced vasodilation Time-to-peak response equal 91
Slower in blacks 35
TEWL Baseline TEWL (in vitro) Higher in blacks 35
Higher in blacks (in vitro) 92
TEWL in response to SLS irritation Higher in blacks and Hispanics 81
(in vivo)
Baseline TEWL (in vivo) Blacks > Caucasians > Asians 93
Return to baseline TEWL after tape Blacks faster than whites 94
Reactivity to SLS (measured by TEWL) Higher in blacks than Caucasians 92
Skin irritant reactivity Reactivity to dichlorethylsulfide (1%) Lower in blacks (measured by erythema, 15% vs. 95
58%) than Caucasians
Reactivity to o-chlorobenzylidene Lower, longer time to response in blacks than 96
malonitrile Caucasians
Reactivity to dinitrochlorobenzene Lower in blacks, but trend towards equalization 97
after removal of stratum corneum than
Reactivity to octanoic acid, 20% SLS, Asians more reactive than Caucasians (react 98
100% decanol, and 10% acetic acid more quickly)
Stinging response Stinging response Lower in blacks than whites 99
Equal in blacks and whites 100
Higher in Asians than whites 33, 101
Skin transparency UV protection factor of stratum corneum Higher in blacks (about 50% higher) than in 102
UVB transmission in stratum corneum Lower in blacks (about 50% lower) 102
Spectral emittance Lower in blacks (above 300 nm: 2–3-fold) 103
UV protection factor of epidermis Higher in blacks (4-fold) 102
UVA transmission through epidermis Lower in blacks (almost 4-fold) 102
UVB transmission through epidermis Lower in blacks (4-fold) 102
Contribution of malpighian layer Black skin: twice as effective in absorbing UVB 102
as white skin
Photoprotection of Skin extensibility on dorsal (sun exposed) Black skin maintains extensibility on sun-exposed 104
epidermis and volar (sun protected) forearms sites, but Hispanic skin extensibility is reduced
on sun-exposed sites
Consequence of Elastic recovery Black skin maintains recovery on sun-exposed 104
photoaging sites, white and Hispanic skins reduced
Drying Higher in Caucasian and Asians than in 105
Hispanics and blacks
Hypertrophic scarring Higher in Asians than Caucasians 106
Response to insult Pigmented dermatoses Higher in Asians than Caucasians 106, 107
Wrinkling Average onset is 10 years later in Asians than 107
Wrinkling Average onset 20 years later in blacks than 108
Thermal tolerance Blacks have a lower threshold than whites 109
Somatosensory Elastic recovery (tested on the cheek) 1.5 times greater in black as compared with white 110, 111
function subjects

Abbreviations: SLS, sodium lauryl sulfate; TEWL, transepidermal water loss; UV, ultraviolet; UVA, ultraviolet band A; UVB, ultraviolet band B.

reactive than Caucasians (11). Asians seemed to be more reac- sensitivities (7). Daily topical use of corticosteroids has been
tive than Caucasians in some studies and less in others, even demonstrated to produce fragile skin (7), and excessive use of
within studies done under the same investigator and protocol topical medications has been demonstrated to be the source of
(25). Tristimulus colorimeter assessment of skin reflectance up to 29% of vulvar dermatitis (38). Drug-induced sensitivity
observed that skin pigmentation was inversely associated is also possible, although no reports on that issue were uncov-
with susceptibility to irritation (14), supported by the find- ered. Interestingly, the thickness of the epidermis in one study
ing that irritant susceptibility to sodium lauryl sulfate (SLS) was demonstrated to be inversely proportional to the number
is decreased after ultraviolet B (UVB) exposure (tanning) (14). of years that the subject had smoked, with a p = 0.0001 (27).
Methyl nicotinate assessment of vasoactive response The specific methodologies and conditions involved in
suggests that there may be genuine racial differences in per- the testing of skin sensitivity introduce a significant amount
meability (35). Increased percutaneous absorption of benzoic of variability into the published results; however, one study
acid, caffeine, and acetylsalicylic acid was demonstrated in reveals that parameters of the testing can themselves induce
Asians when compared with Caucasians, and decreased per- sensitivity apart from that of the specific irritant employed.
cutaneous absorption was observed in blacks (33). Sahlin et al. (39) evaluated the sting potential of the vehicle used
More studies have observed that, while overall preva- in testing the adverse stinging reaction related to lactic acid
lence of skin sensitivity is similar across skin types and ethnic application. The results showed that the ordinary oil-in-water
groups, there are some observable differences with regard to emulsion induced stinging in and of itself; use of a water-in-
what triggers discomfort and how discomfort is experienced. oil emulsion created less discomfort. It was also observed that
Caucasians report visual effects more than African-Americans, decreasing the mineral oil content in the oil-in-water emulsion
while African-Americans are more likely to report sensory resulted in decrease in the degree of sting experienced (39).
effects (26). In addition, African-Americans of both genders
were more likely to report sensitivity in the genital area than Anatomic Site
other groups (p = 0.0008) (3). Differences in skin sensitivity between anatomical regions
A study of 800 women in San Francisco enrolled 200 have been observed. Analysis of structural differences found
subjects in each of four ethnic groups to interview by phone that stratum corneum density varies tremendously by ana-
and found no significant difference in overall prevalence tomical site: palms and soles are the thickest, whereas the geni-
between ethnic groups studied. Euro-Americans, however, tal area is the thinnest (30). The face is the most common site
were found to have a relatively higher susceptibility to wind of skin sensitivity. In a study of 1039 men and women, 77.3%
than other ethnic groups, Asians had significantly higher reported facial sensitivity, compared with 60.7% for the body,
sensitivity to spicy food, and Hispanics had relatively less and 56.2% specifically with regard to genital skin (3). Saint-
reactivity to alcohol (2). Martory also found the face to be the most commonly reported
site of sensitivity, with hands, scalp, feet, neck, torso, and back
Cultural Factors also reported, in order of frequency (6). The nasolabial fold
Cultural factors may play a role. Fastidious cleansing routines has been reported to be the most sensitive region (13) of the
(with douches, perfumes, medication, antifungal medica- facial area, followed by the malar eminence (13), chin, forehead,
tions, and contraceptives), which often precede irritation (28), and upper lip (6). Misery et al. (40) found 44.22% of sensitive-
undoubtedly have some cultural component. Hygiene prac- skin subjects questioned experienced sensitivity of the scalp.
tices are the most common cause of vulvar irritation (28). Factors contributing to facial sensitivity are likely the number
Confounding lifestyle factors should be considered with of products used on the face (particularly in women), a thin-
regard to some observed differences, as cultural practices may ner barrier in facial skin, and a plentitude of nerve endings as
produce widely different exposures to potential irritants (23). well (9). Individual susceptibility appears to be dependent on
For example, older women were found to be more likely to anatomical site (41). Most studies have been conducted in facial
report irritation due to incontinence products than younger skin because of its sensitivity. Stinging sensations, particularly,
women, who were more likely to report irritation due to tam- are readily elicited on facial skin (42). Further, the face is readily
pons (29). Air conditioning is more likely to be reported as a accessible for both visual (43) and biophysical assessments (20).
trigger for sensitive skin in July than in March (36,37). These The vulva is an area of particular interest, since it is
findings are quite likely to be based on increased levels of formed partially from embryonic endoderm; it differs from
exposure than on actual physiological differences. skin at exposed body sites (10). Differences in irritation seem to
be dependent on relative permeability of the irritant in vulvar
Environmental Factors skin; vulvar skin is significantly more reactive than forearm
A majority of sensitive-skin sufferers report unpleasant sen- skin to benzalkonium chloride and maleic acid (44), but less
sory responses to cold temperatures, wind, sun, pollution, and reactive than the forearm to SLS (10). When both venous blood
heat (2,7). An increased susceptibility to SLS was observed in and menses were evaluated for irritant potential, the vulva
the winter compared with the summer (14). Low temperatures was less responsive to both than was the upper arm (45).
and humidity characteristic of winter cause lower water con- Nonkeratinized vulvar skin exhibits clearly increased
tent in the stratum corneum (14). Large-scale epidemiological permeability related to the absence of keratin and loosely
testing in France conducted phone interviews of over 1000 packed, less structured lipid barrier (10). In addition, the
people each in March and then in July, and observed the fre- inner epithelia are thinner, representing a shorter distance to
quency of sensitive skin in women to be significantly higher in penetrate (10). Buccal tissue is often employed in a surrogate
summer than in winter (71.2% in July vs. 59.39% in March) (36). model for vulvar testing, as it has very similar structure and
Numerous other host factors that could influence skin biochemistry (10). Buccal skin has been demonstrated to be 10
sensitivity include unusual occupational or leisure exposures times more permeable than keratinized skin (46).
to chemicals and home climate-control measures (26). Long- Although the vulvar area may be particularly suscep-
term or excessive use of personal-care products can also create tible to cutaneous irritation (47), little objective published data

exists with regard to sensitive skin (42). Irritant reactions to addition, those who believe skin to be sensitive were revealed
feminine-care products have been reported (38) with a few to be more likely to be stingers (59%) than among nonstingers
feminine products that contain chemicals known to be irritants (48.9%) (31).
in certain doses (48). However, the potential for heightened Simion et al., by exaggerated arm-washing with synthetic
vulvar susceptibility to topical agents is not widely reported in detergent bars, observed signs that correlated statistically with
literature (10). The contribution to irritation by topical agents, sensory perceptions (dryness, tightness, and itching). In addi-
though, is substantial (11) and often underestimated (28). In tion, consumers were able to reproducibly distinguish between
fact, 29% of patients with chronic vulvar irritation were dem- test products purely on the basis of sensory effects (50).
onstrated to have contact hypersensitivity, and 94% of those Another study evaluated specific biophysical param-
were determined to have developed secondary sensitization eters in 32 subjects medically diagnosed with sensitive skin in
to topical medications (38). Thus, reported sensitivity in the parallel with a non-sensitive skin control group. Patch testing,
vulvar area may often be related to underlying contact hyper- skin hydration, sebum production, alkali resistance test, lactic
sensitivity because of excessive use of topical hygienic and acid sting test, methyl nicotinate 0.5%, methacholine chloride
medicinal preparations. 1:1000, pH, dermographism, and measurement of total and spe-
Some studies have evaluated skin sensitivity in the cific Ig were performed (19). Patch testing found that patients
vulvar area with regard to sensory responses to consumer with sensitive skin were ten times more likely to respond to
products meant for the vulvar area. It was hypothesized that allergens in the European standard series (p < 0.01) and three
patients with erythema related to a previous genital infection times more likely to respond to cosmetic allergens (p < 0.01)
may represent a population of sensitive subjects; however, no than those without sensitive skin. Sensitive subjects also had
increase in sensory effects to exposure to feminine hygiene significantly less sebum production (p < 0.01) and dryer skin (p
pads was observed (42). < 0.05). Sensitive patients had a four-fold risk of a decrease of
In a similar population, however, in which observed alkali resistance (p < 0.05).
erythema was evaluated against perceived sensory effects, Vascular reactions to methyl nicotinate and methacho-
women who perceived themselves as particularly susceptible line chloride in sensitive-skin patients were observed to be
to facial erythema were significantly more likely to have medi- characterized by a significant hyperreaction of skin blood
cally diagnosed vulvar erythema, a potential indicator of a vessels, with a more intense erythema after methyl nicotin-
underlying biological origin (42). ate application (19). The risk of and intense vascular reaction
Interestingly, a separate study evaluated percep- to methyl nicotinate was 75 times higher in sensitive patients
tions of sensitive skin in women with urinary incontinence, than in non-sensitive subjects, and nearly one-third of sensi-
expecting to observe an increased sensitivity of genital skin tive-skin subjects experienced an abnormal vascular reaction
(49). Increased sensitivity specific to the genital area was not (skin blanching) after application of methacholine chloride.
observed, but incontinent women were significantly more A strong association of sensitivity with fair skin was also
likely to assess themselves as having overall skin sensitivity observed. This may relate to well-established differences in
than continent subjects (p = 0.014: 86.2% in incontinent subjects skin structure and permeability across different skin types.
vs. 68.3% in controls) (49).


It was observed early on that some subjects report a greater ON BIOLOGICAL ORIGIN
incidence of adverse reactions to certain products because of Part of the reason for the observed breakdown between sensory
higher sensitivity (2,32). Some individuals possess exagger- effects and objective signs is the fact that an objective sign like
ated sensitivity to specific individual irritants (34). Despite erythema is the end result of a complex, multistep physiologi-
the fact, however, that studies have demonstrated that sen- cal process. Numerous underlying processes (e.g., changes in
sitive-skin patients are capable of distinguishing products blood flow, moisture content, pH) would be expected to occur
on the basis of blinded sensory endpoints (13,24), a clinically before the appearance of visible external changes (1). A goal
satisfactory description of observed sensitivities remains out of our research has been to increase the ability to predict and
of reach. quantify these subjective consumer responses. Our approach
Tantalizing clues to the underlying mechanisms of has been three-fold: to exaggerate testing conditions to elicit
sensitive skin, however, continue to be reported. If deficits corroborating physical findings, to increase the sensitivity of
in barrier function do play a role in skin sensitivity, regular assessment of physical findings, and to find a way to quantify
use of moisturizer should improve sensitivity; patients who sensory endpoints (1).
completed four daily treatments with moisturizer improved
(8). Evaluation of the potential role of the stratum corneum Exaggeration of Test Conditions
in sensitive skin using corneosurfametry demonstrated that One study evaluated four versions of facial tissues, with and
subjects with demonstrated sensitivity to detergents had an without coating, with repeated wiping to accentuate irrita-
increased reactivity to tested products as compared with the tion (48). Affected skin had been compromised by tape strip-
control group. It may be a specific subgroup of sensitive skin ping prior to wiping protocol initiation. Erythema as well as
with some sort of defect in the stratum corneum that caused dryness were evaluated daily by trained graders. In addition,
weakened resistance to surfactants (18). panelists were interviewed about specific aspects of product
Local anesthetics block response in lactic acid sting tests; preferences. Statistical analysis revealed that the panelists’
stingers respond more vigorously to vasodilators (9). An Italian subjective product preferences were more consistent in distin-
study compared self-reports of sensitivity with response in the guishing between the test products than were either erythema
lactic acid test as follows: stingers were found at very simi- or dryness.
lar prevalences to self-reported sensitivity (56.9% of women A second method of accentuating test conditions, devel-
perceived skin as sensitive, 54.3% revealed to be stingers). In oped in our laboratories specifically for testing paper such as

catamenial products, has proven very effective at accentuating inflammatory response (56). A high precision, handheld infra-
irritant response to inherently mild products. The behind-the- red thermographic scanner makes it feasible to conveniently
knee (BTK) protocol uses the popliteal fossa as a test site and measure local changes in skin temperature in situ (57). Two
adds a relevant mechanical friction component to old testing catamenial products were compared in a BTK protocol. Skin
(51). BTK testing consists of a test product placed behind the surface temperature was measured using an infrared thermo-
knee and held securely by an elastic knee band. Levels of irri- graphic scanner. Subjects were also asked to keep a diary of
tation produced in BTK testing are consistently higher than skin discomfort experienced at test sites, specifically including
those achieved with standard patch testing, and have proven sticking, chafing, burning, itching, pain, edema, or any other
to be consistently reproducible (51). BTK testing, in conjunction issue. Skin temperature changes observed were closely asso-
with the other two approaches below, has proven useful in the ciated with visual scores. In addition, the study incorporated
development of potentially valuable protocols for sensitive- diary-derived data on sensory effects experienced by panel-
skin testing. ists as an additional endpoint. The diaries of subjective sen-
sory experiences over the course of the exposure made a clear
Quantifying Sensory Responses distinction between the two test products that was consistent
A study similar to the facial tissue study above tested femi- with both visual scoring and skin temperatures. A significant
nine hygiene products according to four combinations of t-difference was also observed between mean visual scores
test conditions (wet/dry, intact/compromised skin). Products of those who reported specific adverse sensations as com-
tested were inherently nonirritating and were tested in paral- pared with those who did not report negative sensations. Skin
lel in arm patches and BTK. In addition, the study evaluated temperature means were significantly higher for those who
observed erythema grading against a patient log of sensory reported the adverse sensations rubbing and chafing (interest-
effects. Although no differences were observed between any ingly, burning sensations were not associated with increase
combinations tested, a significant correlation of reported sen- in skin surface temperature). Conditions in this protocol were
sory discomfort with mean irritant scores was observed. Skin optimized for using erythema as the primary endpoint; refin-
sites where patients experienced burning, itching, or sticking ing the protocol to optimize detection of differences in skin
had consistently higher mean irritant scores (52). Ultimately, surface temperature would be a logical next step. Skin surface
eight separate comparison studies were able to statistically temperatures correlated well with visual signs of irritation;
associate perceived sensory effects with an increase in irritant six of eight sensory effects were associated with higher visual
scores (51). scores.
Companion papers that utilized only BTK methodology An additional new technique in development uses
(53) but also evaluated patient diaries in conjunction with the a commercially available product, Sebutape® (CuDerm
irritant testing observed correlation between sensory effects Corporation, Dallas, Texas), an absorbent tape, which is
and mean irritant scores as well (52). applied to skin for 60 seconds and then removed. Application
of the tape to both healthy skin and compromised skin was fol-
lowed by extraction of different cytokines from the Sebutape,
Increasing Sensitivity of Assessment which were then quantified. Levels of IL-1a, IL-1RA, and IL-8
of Physical Response were evaluated. Compromised skin was associated signifi-
Our laboratories evaluated several new methodologies in the cantly with increased IL-1a levels, increased IL-8 levels, and
pursuit of an increased sensitivity of the evaluation of the increased IL-1RA:IL-1a ratio. This technique has not been
physical response. Visual grading of erythema has been relied substantially applied to the problem of sensitive skin yet, but
on for a number of years; trained graders achieve a high degree shows potential (58).
of reproducibility with no specialized equipment (54). A new
approach in our laboratories, however, utilized cross-polar- Links between Sensitive Skin and Immunology
ized light, which allows visualization of the skin at a depth Evidence for a link between atopy and sensitive skin has accu-
of 1 mm below the surface (55). Testing was performed with mulated. An assessment of 1039 individuals (83.6% female)
SLS in a standard, 24-h patch test on the upper arm, and with found that individuals who claimed overall to have sensitive
two different feminine hygiene products (identified as A and skin were 5 times likely to have skin allergies confirmed by a
B) in the BTK. It had been established previously that these doctor (p < 0.0001), and more than 3.5 times more likely to have
two products differed in consumer preference, but no discern- relatives with sensitive skin (59). In a study in older adults,
able difference had been found in objective measures of skin those who claimed sensitive skin had a higher frequency of
effects (24). With the minor irritation produced the SLS patch medically diagnosed skin allergies than younger people who
on the upper arm, both unaided visual scoring and subsur- claimed sensitive skin (29). Loffler et al. (60) observed a link
face visualization detected eythema associated with irritation. between sensitive skin and nickel allergy.
With multiple, shorter exposures (2-h and 6-h) to lower concen- A study compared 25 Greek women with medically
trations of SLS, the subsurface visualization detected eythema diagnosed atopic dermatitis with 25 healthy women (61). A
earlier than unaided visual scoring. Using the mild feminine significant association was found between the clinical diagno-
hygiene products in BTK-enhanced visual scoring through sis of atopic dermatitis and the self-diagnosis of sensitive skin
subsurface visualization allowed the observation of signifi- (p < 0.001). All patients in the atopic dermatitis group described
cant differences in the irritation produced by the two different themselves as having sensitive skin to at least some degree,
products, thus establishing a potential link between sensory with 80% claiming either moderately or very sensitive. By
effects and subclinical irritation. contrast, 64% of individuals in the control group described
A second approach evaluated the potential for changes in their skin as sensitive to some degree, with only 16% claiming
skin temperature related to inflammation to act as a subclinical either very or moderately sensitive. Patients with atopic der-
measure of skin irritation. Previous research has demonstrated matitis were also significantly more likely to indicate a family
a correlation between surface temperature measurements and history of sensitive skin than were non-sensitive individuals

(68%–24%, p = 0.004) 76% of atopic patients who claimed a fam- the myelinated A fiber, A delta fiber, and unmyelinated c-fiber
ily history identified a parent as having sensitive skin. independently (66). In subjects with clinically documented sen-
Atopic individuals were significantly more likely to sitive skin (lactic acid sting test, cosmetic compatibility tests)
report genital sensitivity after contact with hygiene pads, versus non-sensitive controls (all subjects male), nerve fibers
although not more likely to experience sensitivity to genital were stimulated by three different current strengths, and cap-
cleansing products, fragrances, or antiperspirants (61). In addi- saicin (0.075%) was applied to the zygomatic arch. Sensory per-
tion, the study demonstrated a link between clinically diag- ception was verbalized by the subject and recorded. Baseline
nosed atopic dermatitis and sensitive skin, with the frequency, perception of current revealed no significant differences
severity, and history of skin sensitivity in patients with atopic between sensitive and non-sensitive subjects at either 2 kHz or
dermatitis far more pronounced than in controls. This link has 250 Hz, but at 5 Hz—a current known to selectively stimulate
substantial biological plausibility, as contact allergy and skin the c-fibers of sensory nerves. Sensitive skin subjects displayed
sensitivity are phenomena that share similar cytokine induc- a significantly lower perception threshold. In addition, stimu-
tions (59). lation of the skin by capsaicin, in non-sensitive subjects, had
Of potential utility for large-scale screening in industry, no effect on perception of the 5-Hz current, whereas sensitive
postmarket surveillance, and epidemiological testing, a rapid subjects displayed a long-lived increase in the sensory percep-
algorithm containing only three questions has been developed tion threshold (still in place at last time point of 60 minutes).
to quickly identify atopic individuals (62). Testing indicated the These findings imply that sensory perception in sensitive sub-
algorithm was capable of successfully categorizing individuals jects is easily disturbed by weak stimulation, inducing a wide
as “atopic” and “non-atopic” with a 90% success rate (45 out of variability of response compared with non-sensitive subjects,
50 individuals in the test). an effect that appears to be c-fiber modulated. The study was
conducted in only eight subjects (four with sensitive skin) and
should be followed up in a larger population.
Insight into Neurogenic Causality
Sensitive skin is predominantly sensory in nature and thus
ultimately a neurological disorder. Sensory differences may CONCLUSION: A VALID SYNDROME
be related to innervation (63). Dermal nerve fibers extend WITH MULTIPLE ORIGINS?
throughout viable epidermis as free nerve endings, but the Sensitive skin, though now largely recognized as genuine
epidermal component of this network is still poorly charac- syndrome of physiological origin, is still a subjective com-
terized (63). Epidermal nerve density variation could explain plaint with no consistent associations (60), no predictable or
the different sensitivity thresholds in various anatomical classical visible signs of irritation, no immunologically verifi-
sites (64). Hyperreactivity of the neural response of the skin able response, and no accepted and reproducible diagnostic
is postulated to play a role. Possible mechanisms for neu- test (23). Although it is clear that specific individuals clearly
ral system hyperreactivity include nerve fibers; endothelin have heightened sensitivity to different kinds of sensory and
receptors; burn, itch, and heat receptors; cold receptors; and physical irritants, observed reactions are not predictive of gen-
neutrophins (23). eralized sensitivity, and the relationship between observed
Neurogenic inflammation probably results from release sensitivities is unclear (24,67). Evidence suggests that sensitive
of neurotransmitters such as substance P, calcitonin gene- skin may not be a single condition, but the product of multiple
related peptide, and vasoactive intestinal peptide, which etiologies with multiple components. Therefore, the condition
induce vasodilation and mast cell degranulation. Nonspecific may encompass different categories of subjects and sensitivi-
inflammation may also be associated with the release of inter- ties based on different mechanisms (20). Multiple etiologies
leukins (IL-1, IL-8, prostaglandin E2, prostaglandin F2, and would not be farfetched, as the nervous system does not act in
tumor necrosis factor-a) (40). Other studies have evaluated isolation but is interdependent with both the immune system
what contribution neural dysfunction may play in the devel- and the skin, sharing numerous cellular contact as well as the
opment of sensitive skin. same language of cytokines and neurotransmitters. All three
Functional magnetic resonance imaging, which mea- interact to affect cutaneous responses (19).
sured cerebral activation associated with skin discomfort, There is an urgent necessity to establish methodologies
was used to evaluate neural reaction to application of lac- with the capacity to accurately identify sensitive skin (5), inde-
tic acid to the face in 18 women, with and without sensitive pendent of self-assessed reports (23). Methods are needed that
skin (65). Lactic acid–induced skin discomfort resulted in are capable of detecting very subtle skin benefits or potential
increased activity in the primary sensorimotor cortex contra- for adverse effects. Testing has been done primarily on normal
lateral to the application site as well as in a bilateral fronto- subjects, bringing into question the need to focus on examining
parietal network that included the parietal cortex, prefrontal populations that may be inherently more sensitive to irritant
areas around the superior frontal sulcus, and the supplemen- effects (10). Some studies did compare the irritation potential
tary motor activity. In addition, in sensitive skin patients of products between self-declared sensitive skin to non-sensi-
only, group activity spread into the ipsilateral primary sen- tive–skin subjects (68,69). A summary of current methodolo-
sorimotor cortex and the bilateral periinsular secondary gies used to identify sensitive skin is shown in Table 5.4.
somatosensory area, a phenomenon which did not occur in Subclinical irritation may be the key to understanding
the control group. Subjects with self-assessed sensitive skin sensitive skin, as sensations elicited by product exposure are
were also observed to have significantly greater increases in generally discerned long before observable differences (50).
neural activity than those without sensitive skin, demonstrat- One significant advance in the understanding of sensitive skin
ing an increase in neural activity specifically associated with is the development of new, noninvasive techniques; for exam-
sensitive skin. ple, cross-polarized light-enhanced visualization, which has
Another study measured calibrated electrical stimula- demonstrated good correlation with sensory perceptions and
tion of the skin, which stimulates sensory nerve fibers such as the ability to measure subclinical damage (55).

Table 5.4 Some Methodologies Used for Sensitive Skin

Sensory affect Physical effect

Methodology evaluated evaluated Relevant irritants Advantages Disadvantages
Lactic acid (8) Stinging None Cosmetics, other Highly sensitive and Does not predict
personal specific* sensitivity to other
preparations irritants
meant to be left on
Capsaicin (23) Stinging None Cosmetics, other Sensitive, detection Does not predict
personal threshold well sensitivity to other
preparations correlated (inversely) irritants
meant to be left on to perception of
sensitive skin
Sodium lauryl Burning Erythema Industrial exposures, Cheap, quick, reliable Sensitivity to one irritant
sulfate (14) cleaning products assessment of not predictive of
individual general sensitivity,
susceptibility to relationship to sensitive
specific irritant skin in question
Cross-polarized None Subclinical Any potential irritant Permits detection of Requires specialized
light (55) erythema physical changes not equipment
apparent by standard
visual scoring,
Thermoscan (57) None Temperature Any potential irritant Noninvasive, objective, Requires specialized
increases quantitative equipment
resulting from
processes related
to skin injury
Sebutape¹ (58) None Measurement of Any potential irritant Noninvasive, objective, Requires training,
cytokines quantitative, specialized equipment;
produced by potentially very utility for sensitive skin
injured skin sensitive still unassessed

*Lactic acid test positive in 90% of women who claim sensitive skin.

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8. Kligman A. Human models for characterizing “sensitive skin.”
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11. Basketter DA, Wilhelm KP. Studies on non-immune imme-
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Organic Acids with Novel Functions: Hydroxy, Bionic,

N-acetylamino Acids and N-acylpeptide Derivatives
Ruey J. Yu and Eugene J. Van Scott

INTRODUCTION sulfate, heparin, and heparan sulfate. In combination with

Organic acids cover a wide range of organic compounds numerous cosmetic or pharmaceutical agents, hydroxyacids
having an acidic group such as carboxyl, sulfonyl, or phos- have been found to enhance desirable topical effects, and also
phoryl, which include retinoic acid, salicylic acid, benzoic to reduce or prevent side effects caused by topical agents.
acid, and its peroxide form, benzoyl peroxide. The present Because most hydroxyacids are nontoxic, natural, and physi-
discussion will focus on certain organic carboxylic acids ological they are used as primary or secondary ingredients
and related derivatives with unique cosmetic and dermato- in many cosmetic and pharmaceutical products.
logical effects on the skin. These acids are α-hydroxyacids NAAs and NAPs are derived from amino acids or
(AHAs), β-hydroxyacids (BHAs), polyhydroxy acids (PHAs), peptides by substitution at the amino group. N-acetyl-
aldobionic acids (ABAs), N-acetylamino acids (NAAs), and L-cysteine (NAC) has been shown to be a potent anti-
N-acylpeptides (NAPs). oxidant. We have found, for example, N-acetyl-L-proline,
Abnormal keratinization is a principal event associated N-acetyl-L-glutamine, N-acetyl-L-valyl-L-alaninamide,
with a majority of dermatologic conditions including ich- N-acetyl-L-tyrosyl-L-tyrosinamide, and N-acetyl-L-tyrosyl-
thyosis, xerosis, eczema, psoriasis, and acne. As a result, we L-tyrosyl-L-tyrosinamide to be topically effective for reliev-
estimate that over 50% of all skin problems are due to or are ing itch and improving lesions associated with eczema and
associated with disturbed formation and shedding of the stra- xerosis. We have also found that NAAs and NAPs are effec-
tum corneum, and in part attribute the cosmetic and therapeu- tive for topical treatments of aging-related skin changes.
tic uses of AHAs on skin to their unique ability to modulate In this chapter we discuss the scientific basis for topical
the process of keratinization and normalize stratum corneum effects of certain organic acids in dermatological therapy and
exfoliation. in various cosmetic applications.
Hydroxyacids and related acids of nonphenolic origin
are a group of natural and physiological substances which
have profound effects on keratinization and the synthesis of HYDROXYACIDS: NOMENCLATURE
dermal components. Many hydroxyacids and related acids AND OCCURRENCE
occur in food, fruits such as sugar cane, tomato, oranges, Organic hydroxyacids of non-phenolic origin may be clas-
lemons, grapes, apples, mangos, and body tissues. For many sified into the following four groups: AHAs, BHAs, PHAs,
years, cosmetic chemists have used lactic acid along with other and ABAs. For group names, the spellings α-hydroxyacid
organic acids to adjust pH, and citric acid as a chelating and and β-hydroxyacid are preferred instead of α-hydroxy acid and
antioxidant stabilizer in topical formulations. In addition, lac- β-hydroxy acid because α and β indicate the position of hydroxyl
tic acid has been used as a stabilizer in urea formulations for group in the hydroxyacid molecule. In contrast, the spelling
topical treatment of dry skin. polyhydroxy acid is preferred instead of poly-hydroxyacid or
In 1974 the term AHA was first introduced to dermatol- poly-hydroxy acid because in the word, poly indicates many
ogy when it was discovered that AHAs substantially improved or multiple hydroxyl groups, not the position of hydroxyl group
the severe hyperkeratotic conditions of ichthyosis (1). AHAs in the acid (11).
are also beneficial for topical treatment of dry skin, dandruff,
calluses, acne, keratoses, warts, wrinkles, photoaging skin, and
for other cosmetic conditions and dermatological purposes α-HYDROXYACIDS
(2–5). AHAs such as glycolic acid and lactic acid are routinely AHAs are organic carboxylic acids having one hydroxyl
used in peel solutions by estheticians and dermatologists. In group attached directly to the α position of an aliphatic or
dermatological office procedures they are used for topical alicyclic carbon atom, but not to a benzene or other aromatic
management and treatment of various skin conditions includ- ring. On a broader scope, AHAs may include those molecules
ing skin smoothing, acne, and skin changes associated with having additional carboxyl groups (11). Glycolic acid, present
intrinsic and extrinsic aging (6–9). AHAs, on topical applica- in sugar cane juice, is the smallest molecule of all the hydroxy-
tion, have been shown to markedly increase biosynthesis of acids, and is a major ingredient in most AHA products on
hyaluronic acid and collagen in the papillary dermis, although the market. All other AHAs may be considered derivatives
the mechanism of action is unknown (5,10). or substituted glycolic acid. The AHAs may be divided into
Polyhydroxy AHAs constitute one of the two major three subgroups: alkyl AHAs, aralkyl AHAs, and polycar-
components of hyaluronic acid, chondroitin sulfate, dermatan boxyl AHAs.

Table 6.1 Nomenclature and Occurrence of Glycolic Acid and Table 6.2 Nomenclature and Occurrence of Polycarboxy
Alkyl α-Hydroxyacids α-Hydroxyacids
Systematic name Systematic name Common
Chemical structure Common name Occurrence Chemical structure name Occurrence
2-Hydroxyethanoic acid Glycolic acid Sugar cane 2-Hydroxypropane-1,3-dioic acid Tartronic acid
CH2OHCOOH Hydroxyacetic acid HOOC CHOH COOH
2-Hydroxypropanoic acid Lactic acid Tomato 2-Hydroxybutane-1,4-dioic acid Malic acid Apple
2-Methyl Methyllactic acid Mango 2-Methyl-2-hydroxybutane-1,4- Citramalic
2-hydroxypropanoic dioic acid acid
(CH3)2COHCOOH 2,3-Dihydroxybutane-1,4-dioic Tartaric acid Grape
2-Hydroxybutanoic acid α-Hydroxybutyric acid acid
2-Hydroxyoctanoic acid α-Hydroxycaprylic acid 3-Carboxy-3-hydroxypentane-1,5- Citric acid Orange
CH3(CH2)5CHOHCOOH dioic acid
2-Hydroxyeicosanoic acid α-Hydroxyarachidonic C(OH)(COOH) (CH2COOH)2 Lemon
acid 3-Carboxy-2-hydroxypentane-1,5- Isocitric acid
CH3(CH2)17CHOHCOOH dioic acid
2-Hydroxytetraeicosanoic Cerebronic acid Skin as HOOCCHOH CH(COOH)
acid ceramide CH2COOH
CH3(CH2)21CHOHCOOH 3-Carboxy-3-hydroxyhexane-1,6- Homocitric
dioic acid acid
Alkyl AHAs 3-Carboxy-2-hydroxyhexane-1,6- Homoisocitric
A radical attached to the α carbon of glycolic acid can be a dioic acid acid
simple hydrocarbon called alkyl group. The smallest alkyl HOOCCHOH CH(COOH)
group is a methyl group and in this case, the AHA is lactic
3-Carboxy-2-n-hexadecyl-3- Agaricic acid
acid (present in tomatoes). Representative alkyl AHAs are
listed in Table 6.1. 1,5-dioic acid
HOOCCH2 C(OH)(COOH) n-Hexadecyl
Aralkyl AHAs CH(C16H33)COOH citric acid
Aralkyl is an abbreviation of aryl plus alkyl. Aralkyl AHA is
formed when a phenyl group is attached to an alkyl AHA, and
is represented by mandelic acid, benzilic acid, 3-phenyllactic
acid, and atrolactic acid. Mandelic acid has been used in com- POLYHYDROXY ACIDS
bination as methenamine mandelate for oral administration to PHAs are organic carboxylic acids having multiple hydroxyl
treat urinary tract infections. groups (13). Many PHAs are also AHAs; they are derived from
carbohydrates, and are important intermediates in carbohy-
Polycarboxy AHAs drate metabolism. PHAs may be divided into three groups:
AHA may consist of more than one carboxyl group, as aldonic acid, aldaric acid, and alduronic acid.
shown in Table 6.2. Malic acid, occurring in apples, is also
called apple acid, and tartaric acid, present in grapes, has Aldonic Acid
been called fruit acid in the past. Citric acid, occurring in An aldonic acid is a carbohydrate, called aldose, having the
oranges and lemons, has one hydroxyl group and three car- carbon atom at position 1 changed to a carboxyl group, and
boxyl groups. The α- or β-hydroxyacid refers to the position is represented by ribonic acid and gluconic acid, as shown in
of a hydroxyl group as related to a carboxyl group in the Table 6.3. Vitamin C, L-ascorbic acid, is a 1,4-lactone form of
hydroxyacid. When a hydroxyacid has more than one car- the AHA 2,4,5,6-tetrahydroxy-3-ketohexanoic acid, a keto PHA
boxyl group it can be an AHA and BHA at the same time. For with chemical structure: HOCH2 CHOH CHOH CO CHOH
example, malic acid, tartaric acid, and citric acid can be both COOH. The lactone form of vitamin C has two acidic hydroxyl
AHA and BHA. groups at carbon positions 2 and 3, and does not have an effect
on keratinization comparable to that of α-hydroxy PHAs.
BHAs are organic carboxylic acids having one hydroxyl group Aldaric Acid
attached to a carbon atom at the β position, and are represented An aldaric acid is a carbohydrate having two carbon atoms
by β-hydroxybutanoic acid and tropic acid. β-Hydroxybutanoic at the end positions changed to carboxyl groups, and is rep-
acid, also known as β-hydroxybutyric acid, is excreted in resented by glucaric acid (saccharic acid) and galactaric acid
amounts as much as 30 grams per day in the urine of dia- (mucic acid).
betic subjects. Salicylic acid, 2-hydroxybenzoic acid, has both
hydroxyl and carboxyl groups directly attached to a benzene Alduronic Acid
ring. It is not chemically a true BHA, but it is erroneously An alduronic acid is a carbohydrate having the terminal carbon
referred to as a BHA (12) in casual jargon. changed to a carboxyl group, and is represented by glucuronic

Table 6.3 Nomenclature and Occurrence of Aldonic Acids

Systematic name and chemical structure Common name stereoisomer name Occurrence
2,3-Dihydroxypropanoic acid Glyceric acid
3,3-Dimethyl-2,4-dihydroxybutanoic acid Pantoic acid In vitamin B5
2,3,4-Trihydroxybutanoic acid Erythronic acid
HOCH2 (CHOH )2 COOH Threonic acid
2,3,4,5-Tetrahydroxypentanoic acid Ribonic acid, arabinoic acid, xylonic acid, lyxonic acid
2,3,4,5,6-Pentahydroxyhexanoic acid Allonic acid, altronic acid, gluconic acid, mannoic acid, gulonic In skin
acid, idonic acid, galactonic acid, talonic acid
2,3,4,5,6,7-Hexahydroxyheptanoic acid Alloheptonic acid, altroheptonic acid, glucoheptonic acid,
mannoheptonic acid, guloheptonic acid, idoheptonic acid,
galactoheptonic acid, taloheptonic acid

acid which is produced in the body as a detoxifying agent, and Table 6.4 Nomenclature and Source of Aldobionic Acids
forms hyaluronic acid with N-acetyl glucosamine.
Common name Chemical structure Source
Lactobionic acid HOH2 C CHOH Lactose
ABA, also known as bionic acid, consists of one monosaccha-
ride chemically linked through an ether bond to an aldonic
Gal-D-gluconic acid
acid, as shown in Table 6.4. An ABA may also be described as
an oxidized form of a disaccharide or dimeric carbohydrate, Isolactobionic acid 6-O-β-D-Gal-D-gluconic Isolactose
such as lactobionic acid derived from lactose and maltobionic
acid from maltose. Lactobionic acid solution is currently used Maltobionic acid HOH2 C CHOH Maltose
in preservative media for organ transplants. CHOR(CHOH)2 COOH
D-gluconic acid
AHA-RELATED COMPOUNDS Isomaltobionic acid 6-O-α-D-Glc-D-gluconic Isomaltose
An α-ketoacid has a keto instead of hydroxyl group at the acid
alpha carbon of an organic carboxylic acid, and is related to its
Cellobionic acid HOH2 C CHOH Cellobiose
counterpart alpha-hydroxyacid. Pyruvic acid (2-ketopropanoic
acid, CH3 CO COOH) and lactic acid have such a biochemical
relationship in that the latter can be converted to the former R=glucose;4-O-β-D-Glc-
D-gluconic acid
when the hydroxyl group is oxidized to keto group by lactate
dehydrogenase. Gentiobionic acid ROH2 C (CHOH)4 COOH Gentiobiose
D-gluconic acid
Stereoisomers are formed when a carbon in a hydroxyacid has
D-gluconic acid
a stereocenter in the molecule, i.e. the carbon has four non-
Laminarabionic HOH2 C (CHOH)2 Laminarabiose
identical radicals. Whereas glycolic acid, methyllactic acid,
and benzilic acid do not have stereoisomers, lactic acid and
mandelic acid have stereoisomers, D and L forms. Tartronic R=glucose;3-O-β-D-Glc-
acid and citric acid do not have stereoisomers, but malic acid D-gluconic acid
has D and L, and tartaric acid has D, L, and meso forms. For Melibionic acid ROH2 C (CHOH)4COOH Melibiose
PHAs and ABAs, stereoisomers usually result in different R=galactose;6-O-α-D-
chemical names, such as ribonic acid and arabinoic acid, glu- Gal-D-gluconic acid
conic acid and galatonic acid, lactobionic acid and maltobionic Nigerobionic acid 3-O-α-D-Glc-D-gluconic Nigerose
acid, and often retain similar though not identical functions. acid
Sophorobionic acid 2-O-β-D-Glc-D-gluconic Sophorose
Lactone Form acid
In contrast to AHAs and BHAs, many PHAs can form spon-
taneous intramolecular lactones by elimination of water mol-
ecules between the carboxyl and hydroxyl groups, especially δ-lactone, is formed by eliminating one mole of water between
when these two functional groups are separated by two or the carboxyl group and the hydroxyl group at carbon 5 posi-
three carbons. D-Gluconolactone, known as D-gluconic acid tion of D-gluconic acid, forming a six-member ring lactone.

Solubility and Gel Matrix Table 6.5 Biochemical Relationship between Hydroxyacids and
AHAs and BHAs with small molecular weight, and most PHAs Amino Acids
and ABAs, are soluble in water. Certain AHAs and BHAs such Hydroxyacid and amino acid Chemical structure
as methyllactic acid, mandelic acid, malic acid, phenyllactic Glycolic acid CH2OHCOOH
acid, atrolactic acid, and tropic acid are also soluble in alco-
Glycine CH2NH2COOH
hol. Some aralkyl AHAs are lipophilic, and are more soluble in
alcohol than water, such as benzilic acid. Lactic acid CH3CHOHCOOH
One unique property of ABAs is their potential to form Alanine CH3CHNH2COOH
a gel matrix with water. Maltobionic acid can form a clear gel Isopropylglycolic acid C3H7 CHOHCOOH
matrix containing 29% water molecules complexed with mal- Valine C3H7 CHNH2COOH
tobionic molecules. Under the same conditions, lactobionic
3-Isopropyllactic acid C3H7 CH2CHOHCOOH
acid and cellobionic acid can form clear gels containing 14%
and 7% water respectively. The gel matrix may add protective, Leucine C3H7 CH2CHNH2COOH
soothing, and healing effects for inflamed skin or in wound 3-Methyl-3-ethyl-lactic acid (C2H5)CH(CH3)CHOHCOOH
healing. Isoleucine (C2H5)CH(CH3)CHNH2COOH
Glyceric acid CH2OHCHOHCOOH
Acid Strength and pKa Serine CH2OHCHNH2COOH
The acid strength of an organic hydroxyacid is determined
3-Methylglyceric acid CH3CHOHCHOHCOOH
by its proton dissociation from the carboxyl group in aque-
ous solution. After equilibrium is reached, the dissociation Threonine CH3CHOHCHNH2COOH
constant Ka is defined as hydroxyacid anion multiplied by Malic acid HOOCCH2CHOHCOOH
proton ion, and divided by undissociated hydroxyacid based Aspartic acid HOOCCH2CHNH2COOH
on molar concentration. The acid strength is expressed as 3-Phenyllactic acid C6H5 CH2CHOHCOOH
pKa, and the latter is a negative logarithm of the dissociation Phenylalanine C6H5 CH2CHNH2COOH
constant. The hydroxyacid is a stronger acid if its pKa number
is lower (14). The acid strength of a hydroxyacid may not be
related to its topical action on keratinization, although its pKa
is crucial to the determination of bioavailability and bioavail- intermediates for the synthesis of vitamin C in plants and
able concentration. some animals. Citric acid, isocitric acid, and malic acid
are important intermediates in the citrate cycle for energy
Antioxidant Property
Oxidation is defined as removal of electrons or reaction with
oxygen. An antioxidant is defined as any substance capable
Glycosaminoglycans (GAGs) are large carbohydrates widely
of preventing or inhibiting oxidation. In biological systems,
distributed in the body, for example in skin, fibroblasts, mast
an antioxidant may be described as a substance capable of
cells, cartilage, bones, synovial fluid, cornea, and loose con-
disposing, scavenging, or suppressing formation or actions of
nective tissues. Their physiological roles include formation of
peroxide, superoxide, or free radicals. There are three simple
extracellular matrix, specific interactions with collagen and
screen methods which are useful to determine antioxidant
elastin, binding of water and ions, facilitating cell migration,
properties: prevention or retardation of air oxidation of (1)
formation of anticoagulants, and facilitating cell adhesion, cell
anthralin, (2) hydroquinone, or (3) banana peel. Based on
interaction, and cell receptors. There are six different types of
these three tests, all the PHAs and ABAs we have tested are
GAGs, namely hyaluronic acid, chondroitin sulfate, keratan
antioxidants which include ribonolactone, gluconolactone,
sulfate I and II, dermatan sulfate, heparin, and heparan sulfate.
galactonolactone, gulonolactone, glucoheptonolactone, lacto-
Each GAG is formed from two major carbohydrate components
bionic acid, and maltobionic acid (11,15,16). Among AHAs and
which include PHAs. For example, glucuronic acid is one of the
BHAs, citric acid, isocitric acid, tartaric acid, and malic acid
two major components of hyaluronic acid, chondroitin sulfate,
are antioxidants.
and heparan sulfate. Iduronic acid is an important component
of dermatan sulfate and heparin.
Relationship to Amino Acids
Many hydroxyacids are related to or derived from amino acids.
Based on chemical structures, the only difference between an
AHA and an amino acid is the hydroxyl group instead of the
Stratum Corneum Barrier
In normal human skin, the stratum corneum consists of 14 to
amino group, as shown in Table 6.5.
30 layers of corneocytes, including the inner level stratum com-
pactum and the outer level stratum dysjunctum. The keratin-
Carbohydrate Metabolism and Citrate Cycle enriched corneocytes in the stratum corneum are embedded
Many hydroxyacids are intermediate products or end metab- in a lipid matrix and are very resistant to penetration by ionic
olites in carbohydrate metabolism; these include glyceric compounds or large molecules with molecular weight greater
acid in glycolysis. In anaerobic glycolysis, D-glucose is con- than 800 to 1000. While undissociated glycolic acid or lactic
verted to L-lactic acid as the end product. Gluconic acid and acid molecules can readily penetrate into the stratum corneum,
gluconolactone are important intermediates in the pentose the ionized glycolate or lactate anions from the metallic salt
phosphate pathway for the synthesis of nucleotides in DNA cannot. Although the active form of a hydroxyacid may be the
and RNA. Gulonic acid and gulonolactone are carbohydrate anions once inside the skin, a topical formulation must contain

a bioavailable form which can penetrate into and through the therapeutically effective with minimal or no irritation to the
stratum corneum. skin.

Partial Neutralization and Buffered Formulation HYDROXYACIDS: TOPICAL ACTIONS

A topical formulation containing a hydroxyacid without neu- Effects on Keratinization
tralization has a pH below 2. Since the pH of skin surface is On topical application, hydroxyacids exert a profound effect
approximately 4.2 to 5.6, many commercial products contain- on desquamation. At low to moderate concentrations, the
ing glycolic acid or lactic acid are partially neutralized with hydroxyacid, such as glycolic acid 10% cream, on topical
sodium hydroxide or ammonium hydroxide to pH 3.5 to 4.5, application to ichthyotic skin (Figure 6.1) causes initial sepa-
and claim to be buffered formulations. A buffered system is ration of stratum corneum at lower levels near stratum com-
designed to control pH changes of a formulation and does not pactum (3,11). The separation of stratum corneum as a sheet
effectively reduce or eliminate skin irritation without compro- indicates that topical action of the hydroxyacid is not a dis-
mising topical efficacy. Therefore, lessened irritation is mainly persive keratolysis, such as by salicylic acid. A similar event
due to decreased penetration of glycolic acid or lactic acid. can also happen to normal skin. For example, DL-mandelic
acid 10% cream on topical administration twice daily to nor-
Efficacy Potential mal skin causes sudden separation of stratum corneum as a
Cosmetic or therapeutic efficacy of a topical formulation con- thin sheet after a few days of application. The skin exposed
taining a hydroxyacid is proportional to bioavailable concen- after the separation of stratum corneum shows light pink-
tration of the hydroxyacid in an optimal vehicle (13,14). The ish coloration with a shiny smooth surface. With continued
bioavailable concentration is obtained by bioavailability mul- applications such desquamation returns to normal, i.e. can-
tiplied by initial total concentration of the hydroxyacid. The not be perceived. Because of their marked effects on desqua-
bioavailability is defined as a ratio or fraction of the undissoci- mation, hydroxyacids can be topically effective for various
ated hydroxyacid, because only the free acid, not the anion, can cosmetic objectives.
substantially penetrate the stratum corneum. Bioavailability
decreases sharply when the pH is raised. Effects on Dermal Components
and Skin Thickness
HYDROXYACIDS: OPTIMAL Hydroxyacids at concentrations of 10% to 25% on topical appli-
RELEASE FORMULATION cation have been shown to increase biosynthesis of glycosami-
Skin Stinging and Irritation noglycans and collagen fibers, and also to improve the quality
A topical formulation containing a hydroxyacid without par- of photoaged elastic fibers (5,10,18,19). Hydroxyacid 10% to
tial neutralization usually has pH of below 2. Such formulation, 35% creams topically applied twice daily to one forearm and
especially with a small molecular AHA, may provoke sensa- control cream to the opposite forearm for 1 to 9 months have
tions of tingling, itching, stinging, or irritation when applied been found to increase skin thickness very substantially (4,8).
to sensitive, atopic, diseased, or inflamed skin. The undesired The increased skin thickness is mainly due to increased bio-
skin reactions may be due to the lower pH of the formulation, synthesis of glycosaminoglycans and collagen fibers as shown
or uncontrolled release and fast penetration of hydroxyacid by histological analysis (5,19). The degree of increase in skin
into the skin. We have found that faster penetration of an AHA thickness is quite variable, as shown in Table 6.6, and seems
is the major factor in causing skin stinging (17). to depend on individual subject and the type of hydroxyacid
used. In some cases hydroxyacids have been found to increase
Molecular Complex skin thickness more than 40%. Although epidermal thickness
In an amphoteric system, the control-release mechanism is is also increased, the major part of the increase in thickness is
based on intermolecular attracting forces between a hydroxy- the dermis. The increased skin thickness is not due to edema
acid and an amphoteric substance to form a molecular com- formation, because it persists for many weeks to months after
plex. Amino acids are the best amphoteric substances, and discontinuation of topical application. Because of these der-
the preferred ones are arginine, lysine, histidine, trypto- mal effects and increased skin thickness, hydroxyacids are
phan, and ornithine. There are three major attracting forces found to be therapeutically effective for topical treatment of
between a hydroxyacid and an amphoteric substance: ionic/ skin changes associated with aging, including wrinkles and
ionic, dipolar/ionic, and dipolar/dipolar (17). The amphoteric photoaging.
formulations are therapeutically effective with minimal or no In contrast, under the same test conditions salicylic acid
irritations to the skin. at 5% concentration has been found to cause a reduction in skin
In a non-amphoteric system, the control-release mecha- thickness, as shown in Table 6.7. Forearm skin treated with sal-
nism is also based on intermolecular attracting forces between icylic acid clinically appeared thinner.
a hydroxyacid and a non-amphoteric substance to form a
molecular complex. The non-amphoteric substances are multi- Peel Solutions and Skin Peeling
functional organic bases such as amino acid esters, amino acid Certain AHAs can be used in office procedures as peel solu-
amides, aminocarbohydrates, aminoalditols, or aminocyclitols. tions for topical treatment of various cosmetic and dermato-
Examples include glycine ethyl ester, glycinamide, arginin- logical indications, including acne, keratoses, warts, wrinkles,
amide, lysinamide, ornithinamide, glucosamine, glucamine, and photoaging (20,21). The AHAs adaptable for such include
meglumine, and streptamine. In contrast to that of an ampho- glycolic acid, lactic acid, citric acid, and mandelic acid. In wide
teric system, the main attracting force of a non-amphoteric sys- use is glycolic acid in 20, 35, 50, and 70% aqueous solutions
tem is from ionic/ionic force between a hydroxyacid anion and containing small amount of ethanol and propylene glycol for
a cation of a non-amphoteric substance such as glycine ethyl uniform penetration with pH 1.6, 1.3, 1.2, and 0.6 respectively
ester ammonium ion. The non-amphoteric formulation is also (7,8). DL-Lactic acid can be used in the same manner, and 90%

(a) (b)


Figure 6.1 Thirteen-year-old girl with lamellar ichthyosis before (a,b) and after (c,d) topical application of 10% glycolic acid in hydrophilic
ointment twice daily for 3 weeks.

Table 6.6 Increased Skin Thickness by Topical Application of Hydroxyacids and Related Compounds*

Percentage increase
Substance Subject number Age range (Years) Duration (Months) over control
Benzilic acid 2 68–72 2 22–45
Citric acid 13 50–83 5–9 7–55
Glycolic acid 4 58–77 4–8 11–43
Gluconolactone 6 62–81 2–7 7–19
Lactic acid 4 59–70 5–7 17–42
Lactobionic acid 7 49–76 1–3 5–58
Mandelic acid 2 55–62 1 22–27
Methyllactic acid 3 65–76 1–3 14–20
Pyruvic acid 4 62–82 2 14–27

10%–35% Concentration twice daily on forearm skin.


Table 6.7 Decreased Skin Thickness by Topical Application Of and collagen fibers is the capability of these natural and physi-
5% Salicylic Acid Solution Twice Daily on Forearm Skin ological substances to enhance or amplify pharmacologic
actions of many topical agents. Such topical agents include cor-
Subject (Age and Percentage decrease
gender) Duration (Weeks) over control ticosteroids, retinoic acid, hydroquinone, diphenhydramine,
5-fluorouracil, and antifungal agents (11,22). The mechanism
57F 8 –6 of this synergistic action is not known. It may be because
60F 3 –7 hydroxyacids disrupt skin barriers and promote better bind-
59F 6 –8 ing between a topical agent and its receptor molecule, resulting
63F 10 –11 in enhanced topical effect. The enhanced therapeutic effects
61F 2 –12
appear not due to an increased penetration of the topical agent
into the skin. Hydroxyacids can also reduce or eliminate tachy-
60F 5 –14
phylaxis, as well as rebound worsening associated with topi-
67F 6 –21 cal corticosteroids. It has been found that certain side effects
49F 3 –23 associated with topical corticosteroids, such as atrophy, can be
73F 7 –32 reduced or avoided with concomitant use of an AHA (23).


Specificity of Chemical Structure
syrupy liquid having pH 0.5 is commercially available. Citric Unique biological and biochemical actions of a hydroxyacid
acid peel can be used as 20, 30, 40, and 50% aqueous solution depend on specific chemical structure of the molecule, although
with pH 1.5, 1.4, 1.3, and 1.2 respectively. DL-mandelic acid 50% its receptor molecule(s) in the skin has not been identified.
in ethanol solution can be used for light desquamatory peeling. Regarding the three attachment or binding sites, the hydroxyl
Pyruvic acid, an α-ketoacid, is the most powerful peeling group must be neutral and not acidic in chemical property, like
agent but is unsuitable for clinical use because of its chemi- that in alcohol but not like aromatic phenol, which is slightly
cal instability. Glycolic acid 70% solution or DL-lactic acid 90% acidic. The carboxyl group must be attached to a non-aromatic
liquid can provoke epidermolysis on the facial skin, generally carbon, preferably an alkyl chain carbon. The amide or ester
requiring several minutes of exposure depending on skin type. form is substantially less active than the free acid form. The
The clinical sign of epidermolysis is blanching of the skin, side chain can be H, alkyl, or aryl, but the one preferred is a
which indicates the threshold between superficial peeling and short chain. In the case of glycolic acid, the three attachment
deeper peeling. For new patients it is best to begin with 20% or points or binding sites to a receptor molecule(s) in the skin
35% glycolic acid solution and establish a reaction profile for are hydroxyl, carboxyl, and one of the two hydrogen atoms
each subject. In most cases the appearance of erythema is good attached to the alpha carbon. Among related compounds,
indication that the skin will be peeled superficially. The peeling pyruvic acid is the most active and effective α-ketoacid. In con-
process should be terminated by neutralization with sodium trast with the hydroxyacid, the ester form of pyruvic acid, such
bicarbonate solution. The patient may feel mild stinging but as methyl pyruvate or ethyl pyruvate, can be topically active.
degrees of discomfort are mild to moderate and acceptable for It has been speculated that the ester form is hydrolyzed to free
the intended end result. Superficial peeling may be repeated at pyruvic acid by an esterase enzyme in the skin.
intervals of 2 to 3 weeks or longer to provide beneficial effects
in acne, acne-prone skin of younger age, older skin prone to Biological Action
milia and comedones, post-acne scaring, wrinkle-prone skin, The precise mechanisms of actions induced by the hydroxyacid
keratosis-prone skin, and photoaging skin. are not known. Based on the available laboratory and clinical
For superficial peeling with milder reactions, glycolic data, hydroxyacids at different concentrations provide the fol-
acid 50% in ethanol may be used. Application of this solution lowing actions: diminished corneocyte cohesion at lower level
to facial skin is associated with rapid onset of a burning sen- of stratum corneum, near the stratum compactum; a dimin-
sation and erythema. After 1 minute, the skin is rinsed with ished number of desmosomes; reduced epidermal thickness in
sodium bicarbonate solution to relieve the burning sensation. lamellar ichthyosis (Figure 6.2); increased epidermal and der-
In most cases, the erythema fades within 1 to several hours. mal skin thickness in aging skin; increased synthesis of glycos-
Epidermolysis can occur if glycolic acid 50% in ethanol is left aminoglycans and collagen fibers; and increased activities of
on facial skin for several minutes. The erythema may persist dermal dendrocytes (3,5,24).
into the next day, with denudation of skin and degrees of
serous oozing. In some cases, sheet-like separation of stratum
corneum occurs 1 day after the procedure, leaving the skin Biochemical Action
light pink in color and smooth without other overt signs of an Biological actions are usually due to or caused by biochemi-
unwanted reaction. For most cosmetic procedures, superficial cal reactions. In normal stratum corneum, extractable lipids
peeling with 50% or 70% glycolic acid is adequate and can be contain by weight approximately 45% to 50% ceramides, 25%
repeated every 2 to 3 weeks. For deeper peeling, glycolic acid cholesterol, 10% to 15% free fatty acids, and less than 5% each
70% in water or 50% in ethanol can be left on the skin for lon- of other lipids including cholesterol-3-sulfate, which appears
ger periods, and can be so used in the treatment of seborrheic to be involved with cell cohesion in the lower layers of stra-
keratoses and actinic keratoses. tum corneum (25–27). The conversion of cholesterol-3-sulfate
to cholesterol is required for normal desquamation of stratum
corneum in the upper layers (28). It has been shown in X-linked
Synergistic Compositions ichthyosis that the skin is deficient in steroid sulfatase enzyme
Associated with the ability of hydroxyacids modulating kera- (29). While cholesterol is non-ionic, cholesterol-3-sulfate is an
tinization and inducing biosynthesis of glycosaminoglycans ionic compound which may cause stronger intercorneocyte

(a) (b)

Figure 6.2 Two-year-old girl with lamellar ichthyosis, including scaly skin and erythema, before (a) and after (b) twice daily topical appli-
cation of 8% gluconolactone in control-release combination formulation for 4 weeks.

binding and cohesion, resulting in retarded desquamation. We cohesion and dislodge early comedones from follicular orifices
might speculate that the hydroxyacid activates steroid sulfatase (6). A gel or solution formulation in water, ethanol, propylene
to enhance hydrolysis of cholesterol-3-sulfate to free choles- glycol 40:40:20 ratio containing 5% to 10% hydroxyacid and
terol in the stratum compactum of ichthyotic skin. AHAs such applied twice daily is usually quite beneficial for topical treat-
as glycolic acid, lactic acid, and citric acid have been shown to ment or prevention of early acne. The hydroxyacids include
activate factor XIIIa transglutaminase enzyme, tumor necrosis glycolic acid, lactic acid, methyllactic acid, mandelic acid, and
factor-α, and to stimulate mast cells, fibroblast cells, and der- benzilic acid. In general, improvement of acne lesions is dis-
mal dendrocytes. cernible within a few weeks of starting topical treatment.
For moderate to severe acne, hydroxyacids at higher skin
HYDROXYACIDS: COSMETIC AND peeling concentrations can be used to cause epidermolysis to
DERMATOLOGICAL INDICATIONS unroof pustules and beneficially modulate follicular epithe-
Dry Skin and Skin Smoothing lium to the level of sebaceous glands (7,33). Glycolic acid 50% or
Hydroxyacids can modulate keratinization at the levels of the 70% aqueous solution containing small amounts of alcohol and
stratum compactum, and such action is desirable for topical propylene glycol can be effectively used as peeling solution.
treatment of dry skin conditions. Most cosmetic products for dry The solution is applied to acne-involved areas with a cotton
skin contain humectants or moisturizers which tend to improve ball or suitable brush. The patient will feel a mild to moder-
water content or prevent water loss from the stratum corneum. ate sense of burning as erythema develops over a period of a
Although lactic acid has been claimed to be a moisturizer, most few minutes. When skin blanching or perifollicular edema is
AHAs are not primary humectants or moisturizers. Rather, they detected, the skin is neutralized with 5% sodium bicarbonate
modulate keratinization to normalize or improve the quality of solution to stop further epidermolysis. With this procedure,
stratum corneum so that water loss is minimized. Most AHAs most pustules will become unroofed. The procedure may be
and BHAs, 4% to 10% cream or lotion, are therapeutically effec- repeated every 2 to 3 weeks. During intervening periods, a low
tive for topical treatment or prevention of common dry skin or concentration such as 5% to 8% hydroxyacid in gel or solution
xerosis (30,31). PHAs and ABAs 5% to 10% concentration have may be used by the patient once or twice daily to keep follicles
extra benefits for dry skin because of multiple hydroxyl groups opened.
in the molecule, which seem to bind water molecules through Rosacea is characterized by vascular dilatation with
hydrogen bonding. In addition, these hydroxyacids are gentle to erythema near the center of the face. The cause is unknown,
sensitive or inflamed skin without causing skin stinging. although sunlight may play an important role in the develop-
Hydroxyacids such as glycolic acid, lactic acid, and man- ment of rosacea lesions. Rosacea lesions can evolve into telan-
delic acid at 10% concentration are therapeutically effective for giectasia with acneiform papules and pustules, and the skin is
topical treatment of ichthyosis and other severe dry skin con- quite sensitive to many topical agents. Metronidazole 0.75% gel
ditions. PHAs and ABAs such as gluconolactone, lactobionic is beneficial for topical treatment of rosacea but has no effect
acid, and maltobionic acid 10% to 15%, alone or in combina- on telangiectasias. Because PHAs and ABAs are antioxidants
tion with other topical agents, are beneficial and soothing for and gentle to sensitive skin, substances such as gluconolactone,
topical treatment of eczema and psoriasis (58). Combination of lactobionic acid, and maltobionic acid at 5% to 10% concentra-
several hydroxyacids seems to be the best for topical treatment tions are beneficial for prophylactic as well as topical treatment
of severe dry skin. of rosacea (34).

Acne and Rosacea Warts (Verrucae Vulgares)

All acne lesions involve retention of follicular stratum cor- Hydroxyacids are therapeutically effective for topical treat-
neum. Hydroxyacids are therefore therapeutically effec- ment of warts caused by human papillomavirus, which induces
tive for topical management of acne (32). Topical action of a extreme degrees of hyperkeratosis (6). A rational approach
hydroxyacid at lower concentrations can diminish corneocyte to topical treatment includes removing the hyperkeratotic

“armor,” destroying the tissue harboring the virus, and intro- Extrinsic aging is a combination of intrinsic aging and
ducing antiviral agent(s). accelerated degeneration caused by ultraviolet (UV) radiation,
Much of the hyperkeratotic plate can be removed by scal- ionizing radiation, air pollution, wind, cold, heat, dampness,
pel paring. Thereupon an AHA in 70% solution or gel can be chemicals, smoke, and cigarette smoking. Face and hands are
applied under occlusion twice daily by patients for a week or typical skin areas showing extrinsic skin aging. Photoaged
more to cause epidermolysis. In most cases, destruction of wart skin is rough, dry, mottled, yellowish, leathery, and thickened.
tissue occurs by epidermolysis and is sufficient to eradicate the It lacks elasticity, has keratoses and pigmented age spots and
virus as well as the lesion. However, combined use of an anti- coarse and deep wrinkles. Aging of the face and the hands in
metabolite such as 5-fluorouracil (5-FU) is more curative, and elderly people is due to a combination of intrinsic aging and
it can be applied concomitantly (7). For home use by patients, photoaging. Physiological aging cannot be stopped, but signs
0.5% 5-FU solution is prepared by dissolving the drug in 70% of extrinsic cutaneous aging can be modified by topical appli-
glycolic acid. The solution is applied twice daily with a cotton cation of AHAs, PHAs, ABAs or combinations to improve
applicator to the center of the wart, which is then covered with appearance and slow the process (39–42) (Figure 6.3).
tape. Applications are discontinued if discomfort occurs and
resumed if the wart lesion is not yet resolved. We have found Sunburn Cells
that this treatment usually results in complete resolution of Sunburn cells are dead or dying keratinocytes caused by UV
lesions within 3 to 4 weeks. radiation, and are physiologically programmed for cell death
(apoptosis). The cells are eosinophilic and appear in the epider-
Eczema and Pruritus mis within 30 minutes, and are maximal at 24 hours after UVB
Eczema may be defined as persistent inflammatory skin radiation (290–320 nm) (43). The action spectrum appears to be
lesions with constant or repeated itch. Eczematous disorders just below 300 nm, and UVA (320–400 nm) radiation does not
can occur at any age and in various forms, such as nummular seem to produce any detectable or significant numbers of sun-
eczema and lichen simplex chronicus, and is a common skin burn cells in epidermis. The mechanism of action is unknown.
disease in Asian countries. Eczema may be caused by endo- It appears that DNA damages have occurred in certain kera-
geneous and exogeneous factors. Pruritus is the main distur- tinocytes at the lower epidermis after the UVB radiation, and
bance. Corticosteroids have been used for topical treatment of these sunburn cells move rapidly upward through the epider-
eczema, and the pruritus diminishes secondarily as the inflam- mis into the stratum corneum.
matory process is subdued. In cases of eczema wherein inflam- Preliminary study showed that glycolic acid 10%, pH 3.5,
mation is not a primary event, topical corticosteroids are not on topical application for 12 weeks decreased minimal ery-
very effective in eradicating the pruritus. Hydroxyacids such thema dose by 18% and increased the number of sunburn cells
PHAs and ABAs incorporated into topical antipruritic formu- by twofold, as compared to control group (44). Under the same
lations can greatly enhance efficacy. For example, addition test conditions, a cosmetic surfactant, sodium lauryl sulfate
of gluconolactone, lactobionic acid, and/or maltobionic acid at 0.5% concentration, increased the number of sunburn cells
to diphenhydramine topical formulations greatly enhances sixfold. Topical formulations containing an AHA and a sun-
antipruritic efficacy. The best results are obtained when gluco- screen agent with a sun protection factor (SPF) approximately
nolactone, lactobionic acid, and/or maltobionic acid are com- 3–4 appeared to prevent any increase of sunburn cells (45).
bined with hydrocortisone-17-valerate and diphenhydramine Topical formulations containing a PHA, 5% to 10% gluconolac-
in cream or lotion vehicles. tone, without any sunscreen agent have been shown to provide

Onychomycosis is a paramount cosmetic affliction, heretofore
difficult to treat. When an AHA is incorporated into a compo-
sition containing an antifungal drug, the formulation becomes
topically very effective for nail infections. Improvement of
fingernail and toenail infections progresses at the rate of nail
growth; approximately 1 mm per week for a fingernail and 0.5
mm per week for a great toenail. The improvement rates also
suggest that fungal infection of the nail is arrested by topical
treatment with synergistic compositions containing both AHA
and antifungal drug. Fungal infections of fingernails have been
regularly eradicated with up to 6 months of topical treatment
with solutions containing 2% clotrimazole and 20% glycolic acid.


Cutaneous aging is caused by internal and external factors
(35–38). Intrinsic aging is a physiological degeneration caused
(a) (b)
by declining ability and functions inherent with increasing
age. Upper arms and buttocks are typical skin areas showing
intrinsic aging, where the skin thins and develops fine wrin- Figure 6.3 Fifty-five-year-old woman with photoaged skin,
kles. Daily topical application of glycolic acid, gluconolactone, including coarse wrinkles and textural signs of elastosis on her
or lactobionic acid 5% to 10% cream or lotion has been found face, before (left) and after (right) 70% glycolic acid peels monthly
to be beneficial for prophylactic as well as topical treatment of and home use of twice daily 10% glycolic acid cream for 9 months.
sun-protected areas of aging skin.

therapeutic effects and protect the skin from any increase in

sunburn cells (46).
Nevertheless, regular use of a sunscreen is advisable.

Actinic Keratoses
Actinic keratosis is a precancerous lesion of keratinocytes, also
called solar keratosis, which is caused by photodamage. The
lesions are on the sun-exposed areas of skin. Actinic keratosis
may progress to squamous cell carcinoma. One conventional
treatment is continued topical application of 5-FU for several
weeks. A combination of a hydroxyacid and 5-FU can shorten
the treatment time and the period of discomfort from several
weeks to just 1 week. First, the lesions or sites of actinic kera-
toses are identified by topical application of 5% 5-FU cream
twice daily to affected areas for 5 to 7 days before the office
procedure. Once the lesions are identified, 70% glycolic acid in
ethanol:propylene glycol 80:20 is applied to the lesions. After 2 (a) (b)
to 5 minutes when the lesions begin to blanch, 5% 5-FU solu-
tion is applied.
Alternatively, the lesions may be treated with 0.5% to Figure 6.4 Eighty-year-old woman with photodamaged skin,
including coarse wrinkles and multiple lesions of actinic keratoses
1% 5-FU dissolved in 30% glycolic acid aqueous solution (7).
on her face, before (left) and after (right) 70% glycolic acid peels
In most cases, this procedure results in complete eradication and home use of 10% glycolic acid cream for 4 years.
of the lesions.

Age Spot Keratoses

Aging-related macules and papules on the face and the back of
the hands are pigmented lentigines, non-pigmented keratosis,
and/or seborrheic keratoses. For rapid removal of keratoses,
100% pyruvic acid, 90% lactic acid, or 70% glycolic acid peel solu-
tions may be used as an office procedure (Figure 6.4). After the
area is degreased with 70% ethanol, the peel solution is applied
with a fine camel-hair brush, and the skin is neutralized with
5% sodium bicarbonate solution when epidermolysis occurs.
Among the above peel solutions, 70% glycolic acid is preferred.
The skin peel may be repeated after an interval of several weeks
to eradicate remaining lesions. Home treatment with a cream or
gel containing 10% hydroxyacid with or without 2% hydroqui-
none may be continued to subdue re-emergence or new lesions.
Age spots on the dorsa of the hands and forearms, more resis-
tant to simple topical treatment, may need sustained treatment
with 20% or higher concentration of hydroxyacid (7). The addi-
tion of hydroquinone seems more effective in the eradication
of pigmented skin spots such as lentigines and freckles (Figure (a) (b)
6.5). The time required for their clinical resolution is variable,
from a few months to a year or more. Figure 6.5 Sixty-three-year-old man with age spots, including
multiple lesions of seborrheic keratoses and lentigines on his left
Wrinkles and Aging Skin face, before (left) and after (right) twice daily topical application
of 10% glycolic acid and 2% hydroquinone cream for 21 months.
AHAs are therapeutically effective for topical treatment of
skin changes associated with aging, because they increase
skin thickness by stimulating biosynthesis of GAGs and col-
lagen fibers. An AHA such as glycolic acid at concentration of
10% can be used by a patient at home to treat fine wrinkles Antioxidants and Photoaging
on the face (6,7). Substantial improvement may be perceived The human body needs oxygen for energy and life itself.
after several months of treatment. However, the time required Therefore, oxidation is inherent to living. Reactive oxygen
for clinical improvement of coarse wrinkles may take years, species (ROS) are produced by oxidation or peroxidation, and
depending on the degree of severity. Office procedures using include superoxide, hydrogen peroxide, hydroxyl radicals,
90% lactic acid or 70% glycolic acid peels seem to provide faster and peroxyl radicals (48,49). Among these ROS, the hydroxyl
resolution of coarse wrinkles, combined with sustained home radicals are most reactive and cause cellular and tissue stress
use of an AHA at or near 10% concentration (8,40,47,57,59). by reacting with proteins, nucleic acids, lipids, and other bio-
Five parameters may be used to monitor the progress of chemical entities. Under quiescent conditions, endogenous
therapeutic effects: improvement in dyspigmentation, skin tex- or available antioxidants and reductive enzymes in the body
ture, overt fine lines, overt wrinkles, and increased skin thick- are capable of neutralizing these harmful ROS. Known anti-
ness. It is essential that “before” and “after” photos be taken for oxidants and enzymes include vitamin C, vitamin E, reduced
each patient to assess progress. α-lipoic acid, reduced ubiquinones (coenzyme Q), reduced

glutathione (GSH), reduced nicotinamide adenine dinucleotide and ABAs, such as gluconolactone and lactobionic acid, are
phosphate (NADPH), and superoxide dismutase, which con- used as unique ingredients in cosmetic products.
verts superoxide to hydrogen peroxide and oxygen. Catalase
and glutathione peroxidase in turn convert hydrogen peroxide Antagonistic Acetoxyacids
to water and oxygen. Many other antioxidants probably have Like some substances with antagonists in nature, hydroxy-
ancillary or primary roles. acids appear to have their own antagonistic counterparts.
Sunlight is essential for life on earth, but UV radiation is When the hydroxyl group at the α position of an AHA is
harmful to human skin. Human skin is equipped with antioxi- acetylated to an acetoxyl compound, the modulation on kera-
dant systems that counteract or dispose of some ROS induced tinization changes to reverse direction, causing hyperkera-
by UVB (50). However, the amount of these endogenous or tinization. The antagonistic action is noticeably pronounced
available antioxidants may not be sufficient to overcome the when an aralkyl AHA such as mandelic acid, benzilic acid,
increased ROS produced by continued exposure to UVB. The or phenyllactic acid is acetylated to O-acetyl-mandelic acid,
skin reactions or damages caused by UVB include erythema, O-acetyl-benzilic acid, or 0-acetyl-phenyllactic acid, respec-
edema, exfoliation, tanning, abnormal thickening (elastosis) tively. Aralkyl O-acetyl-AHA 5 to 10% creams have been
or thinning of the epidermis and dermis, and numerous other shown to increase thickness and compactness of stratum cor-
changes known as photoaging, including carcinogenesis. neum in hairless mouse and human forearms (3). The hyper-
Hydroxyacids have been shown to improve the appear- keratotic action of aralkyl O-acetyl-AHAs has been found to
ance of photoaged skin by improving epidermal renewal be useful and effective for topical treatment of brittle nails,
and desquamation and by increasing dermal biosynthesis of psoriatic nails, and cheilitis caused by oral administration of
GAGs and collagen fibers (5,10). Because PHAs and ABAs are 13-cis-retinoic acid.
antioxidants, they can be used to prevent or counteract ROS
induced in the skin by UVB, in conjunction with sunscreens N-Acetylamino Acids and N-Acetyl Compounds
and sunblocks in cream, lotion, or gel form to prevent sun An amino acid is an organic acid having one or more than one
damage (34). alkaline radical such as amino, guanidino, imino, or hydrazine
radical attached at any carbon atom other than carbon one, and
SIMILARITIES AND DIFFERENCES NAA is obtained by N-acetylation of the amino group. In fact,
OF HYDROXYACIDS the NAA can be considered as the organic acid in which one
The chemical structure of a hydroxyacid determines whether H is replaced by an acetamino group. There are 20 common
the substance belongs to AHA, BHA, PHA, or ABA, and such amino acids present as L form in natural proteins, and there are
classification is also based on its characteristics and usage. also a number of related amino acids with different chemical
Different members of the same group may possess different structures and configurations. These common amino acids and
physicochemical properties, e.g., hydrophilic or lipophilic, related amino acids can form NAAs and are related N-acetyl
but they have similarities in their topical actions with differ- compounds. N-Acetylglucosamine, N-acetylgalactosamine,
ent degrees of potency; these include glycolic acid, lactic acid, and N-acetylmannosamine are N-acetylated derivatives
and mandelic acid. We have found that BHAs as a group are of aminocarbohydrates glucosamine, galactosamine, and
similar to AHAs in many ways, and the same is true between mannosamine, which are organic aldehydes instead of
ABAs and PHAs. However, PHAs and ABAs are functionally organic acids. Topical actions of N-acetylglucosamine and
different from AHAs and BHAs in certain aspects, as shown N-acetylgalactosamine have some similarities to that of NAA,
in Table 6.8. Because of multiple hydroxyl groups in the mol- as shown in Table 6.9. We have found that N-acetyl-L-proline
ecule, PHAs and ABAs are antioxidants, are gentle to the skin, and N-acetyl-D-glucosamine at 5% to 10% concentration are
and they do not increase sunburn cells following UV radiation. topically effective for ichthyosis and also for eradication of itch
Certain members of PHAs, such as glucuronic acid and idu- associated with eczema and xerosis.
ronic acid, are known constituents of GAGs. In the past, AHAs, Some NAAs and N-acetylaminocarbohydrates occur
especially glycolic acid, have been used quite extensively for in nature as metabolites, biopeptides, glycoproteins, or gly-
cosmetic and dermatologic indications. More recently, PHAs cosaminoglycans, e.g. N-acetyl-L-glutamic acid in liver (51),

Table 6.8 Similarities And Differences In Characteristics And Use Of Hydroxyacids

Characteristics/Use AHAs BHAs PHAs ABAs

Physiological nutrients or natural substances + + + +
Antioxidants against superoxides, free radicals * + +
Gentle to sensitive skin + +
Gel matrix formation/wound-healing +
Constituents of GAGs +
Modulate keratinization, dry skin, acne, keratosis + + + +
Increase dermal components GAGs, collagen, elastin + + + +
Reduce wrinkles, photoaging + + + +
Synergistic effects of corticosteroids, antifungal agents + + + +

Abreviations: AHAs, α-hydroxyacids; BHAs, β-hydroxyacids; PHAs, polyhydroxy acids; ABAs, aldobionic acids; GAGs, glycosaminoglycans
*Polycarboxy AHAs; malic acid, citric acid, tartaric acid are antioxidants

Table 6.9 N-Acetylamino Acids and N-Acetyl Compounds

Topical effects
Chemical name Chemical structure Ichthyosis Itch
N-Acetyl-L-alanine CH3 CH(NHCOCH3) COOH
Nα-Acetyl-L-arginine H2NC(=NH)NH(CH2)3 CH(NHCOCH3) COOH
N-Acetyl-L-aspartic acid HOOC CH2 CH(NHCOCH3) COOH
N-Acetyl-DL-asparagine H2NOC CH2 CH(NHCOCH3) COOH 4+
N-Acetyl-L-cysteine HSCH2 CH(NHCOCH3) COOH 3+
N-Acetyl-glycine CH2(NHCOCH3) COOH 3+
N-Acetyl-L-glutamic acid HOOC CH2 CH2 CH(NHCOCH3) COOH 4+
N-Acetyl-L-glutamine H2NOC CH2 CH2 CH(NHCOCH3) COOH 4+
N-Acetyl-L-histidine C3H3N2 CH2 CH(NHCOCH3) COOH
N-Acetyl-L-isoleucine CH3 CH2 CH(CH3) CH(NHCOCH3) COOH
N-Acetyl-L-leucine (H3C)2CH CH2 CH(NHCOCH3) COOH
Nα-Acetyl-L-lysine H2NCH2 (CH2)3 CH(NHCOCH3) COOH 4+
N-Acetyl-L-methionine (H3C)SCH2 CH2 CH(NHCOCH3) COOH
N-Acetyl-L-phenylalanine C6H5 CH2 CH(NHCOCH3) COOH
N-Acetyl-L-proline N(COCH3)C4H7 COOH 4+ 4+
N-Acetyl-L-serine HOCH2 CH2(NHCOCH3) COOH
N-Acetyl-L-threonine H3C CHOH CH(NHCOCH3) COOH
N-Acetyl-L-tryptophan C8H6N CH2 CH(NHCOCH3) COOH
N-Acetyl-L-tyrosine HOC6H4 CH2 CH(NHCOCH3) COOH 4+
N-Acetyl-L-tyrosinamide HOC6H4 CH2 CH(NHCOCH3) CONH2 4+
N-Acetyl-L-valine (H3C)2CH CH(NHCOCH3) COOH
N-Acetyl-β-alanine CH2(NHCOCH3) CH2 COOH 4+
N-Acetyl-γ-aminobutanoic acid CH2(NHCOCH3) CH2 CH2 COOH 3+
Nα-Acetyl-L-ornithine H2NCH2 (CH2)2 CH(NHCOCH3) COOH
N-Acetyl-L-citrulline H2NCONH(CH2)3 CH(NHCOCH3) COOH
N-Acetyl-creatine H2NC(=NCOCH3)N(CH3) CH2 COOH
N-Acetyl-creatinine -HNC(=NCOCH3)N(CH3) CH2 CO-
N-Acetyl-phenylglycine C6H5 CH(NHCOCH3) COOH
N-Acetyl-4-hydroxyphenylglycine HOC6H4 CH(NHCOCH3) COOH 3+
N-Acetyl-D-glucosamine HOH2C (CHOH)3 CH(NHCOCH3) CHO 4+ 4+
N-Acetyl-D-galactosamine HOH2C (CHOH)3 CH(NHCOCH3) CHO 4+

Ichthyosis: 3+, 75%; 4+, 100% improvement

Itch: 4+, eradicate itch completely for 8 hours

N-acetyl-L-aspartic acid in brain (52), N-acetyl-L-serine in N-Acylpeptides

melanocyte-stimulating hormone (α-MSH) (53), N-acetyl- NAP is an acylated peptide derivative. A peptide is formed
L-tyrosine in N-acetyl-β-endorphin (53), N-acetyl-D- from two or more amino acids by a covalent amide bond,
glucosamine in hyaluronic acid and keratan sulfate (54), and C(=O)NH, when the carboxyl group on one amino acid reacts
N-acetyl-D-galactosamine in chondroitin sulfate and derma- with the amino group of the other amino acid in a dehydration
tan sulfate (54). reaction. A dipeptide is formed from two amino acids, a tri-
NAC is a potent antioxidant against free radicals such peptide is formed from three amino acids, and a polypeptide
as hydroxyl radical (55) and is a good precursor for glutathi- is formed from multiple amino acids. The 20 common amino
one synthesis in the body. NAC is used as a mucolytic, detoxi- acids are represented by chemical names, such as “glycine,” or
fying, and antiviral agent. We have found that NAC 8 % in abbreviated symbols such as three letters, “Gly,” or one letter,
hydrophilic ointment is topically effective for ichthyosis “G.” Except for glycine, all other common amino acids have
(Figure 6.6). stereoisomers, i.e., enantiomer, D, or L form. The amino acids
An amino acid can be in amide or hydrazide form, e.g. in most natural peptides and proteins are all in L-form.
N-acetylaminoamide and N,N'-diacetylaminohydrazide. We The three-letter symbols used for the 20 common amino
have found that N-acetyl-L-tyrosinamide and N,N'-diacetyl- acids are as follows: alanine (Ala), arginine (Arg), aspartic acid
L-tyrosinhydrazide in oil-in-water emulsion on topical appli- (Asp), asparagine (Asn), cysteine (Cys), glycine (Gly), glutamic
cation to normal human skin can stimulate biosynthesis of acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leu-
hyaluronic acid, and increase skin thickness. cine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe),

(a) (b)

Figure 6.6 Six-year-old boy with lamellar ichthyosis before (a) and after (b) topical application of 8% N-acetyl-L-cysteine in hydrophilic
ointment twice daily for 4 weeks.

proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), differences in topical actions. Both salicylic acid and glycolic
tyrosine (Tyr), and valine (Val). acid have similar beneficial effects on disturbed keratiniza-
Common acyl groups are acetyl (Ac) and propanoyl (Pa) tion, e.g. acne, ichthyosis, calluses, etc. Glycolic acid increases
radicals, such as N-acetyldipeptide and N-propanoyldipeptide.
The NAP derivatives can have different forms at carboxyl Table 6.10 N-Acylpeptide Derivatives
terminal group such as free acid (OH), amide (NH2), ethyl Aging related Itch,
ester (OEt), hydrazide (NHNH2), and N-acetyl hydrazide N-acylpeptide derivatives skin changes eczema
N-Ac-L-Ile-L-Ala-NH2 2+ 4+
NAP derivatives at concentrations of 0.5% to 10% on
topical application have been found to increase skin thick- N-Ac-L-Ieu-L-Ala-NH2 3+ 4+
ness. For example, N-acetyldipeptide amide, N-Ac-L-Tyr-L- N-Ac-L-Ieu-L-Ala-OH 2+ 3+
Tyr-NH2, at 3% increased skin thickness 15% to 20% after N-Ac-L-Val-L-Ala-NH2 4+ 4+
4 weeks; N-acetyltripeptide amide, N-Ac-L-Tyr-L-Tyr-L- N-Ac-L-Val-L-Ala-OH 2+ 4+
Tyr-NH2, at 0.5% increased skin thickness 5% to 10% after
N-Pa-L-Val-L-Ala-OH 2+ 4+
7 days; N-acetyltripeptide ethyl ester, N-Ac-L-Tyr-L-Tyr-L-
N-Ac-L-Cys-L-Cys-NH2 3+ 2+
Tyr-OEt, at 0.5% increased skin thickness 20% to 30% after
10 days. The increased skin thickness or plump was not due N-Ac-L-Cys-L-Cys-OH 3+ 2+
to increased water retention or edema of the skin, because N-Ac-L-Ile-Gly-NH2 3+ 4+
the thickness was maintained for many months after dis- N-Ac-L-Ile-Gly-OH 2+ 4+
continuation of the treatment. As shown earlier (5) increased N-Ac-L-Ieu-Gly-NH2 3+ 4+
skin thickness was due to increased biosynthesis of GAGs
N-Ac-L-Ieu-Gly-OH 2+ 3+
and collagen fibers, improved quality of elastic fibers, less
clumping of melanin, and lighter appearance of age spots. N-Pa-L-Ieu-Gly-OH 2+ 3+
Therefore, increased skin thickness is expected to improve N-Ac-L-Pro-Gly-NH2 2+ 4+
aging-related skin changes, including fine lines, wrinkles, N-Ac-L-Val-Gly-NH2 2+ 4+
photoaging, age spots, blotches, hyperpigmented skin, mot- N-Ac-L-Val-Gly-OH 2+ 4+
tled skin, and can be used for younger-looking skin and skin N-Ac-L-Ala-L-Ile-NH2 2+ 3+
lightening (Table 6.10).
N-Ac-L-Ile-L-Ile-NH2 3+ 4+
NAP derivatives at concentrations of 0.5% to 5% on topi-
cal application have been found to eradicate itch within a few N-Ac-L-Cys-L-Tyr-NH2 2+ 3+
minutes and improve eczema lesions within a few weeks with N-Ac-L-Tyr-L-Tyr-NH2 4+ 4+
continuing topical application (Figure 6.7). N-Ac-L-Tyr-L-Tyr-OH 4+ 4+
N-Ac-L-Tyr-L-Tyr-NHNH2 4+ 2+
Organic acids cover various organic compounds which include N-Ac-L-Tyr-L-Tyr-L-Tyr-OH 2+ 3+
retinoic acid, salicylic acid, α-hydroxyacids (AHAs), polyhy- N-Ac-L-Tyr-L-Tyr-L-Tyr-OEt 3+ 4+
droxy acids (PHAs), aldobionic acids (ABAs), N-acetylamino N-Ac-L-Tyr-L-Tyr-L-Tyr-NH2 4+ 4+
acids (NAAs), and N-Acylpeptides (NAPs). On topical admin- N-Ac-L-Val-L-Val-L-Ala-NH2 2+ 3+
istration, these organic acids exert distinctive pharmacological N-Ac-L-Tyr-L-Val-L-Tyr-NH2 3+ 4+
actions on keratinization and/or biosynthesis of dermal com-
ponents; i.e. glycosaminoglycans, collagen, and elastic fibers. Note: Aging skin including age spots; itch and eczema: 2+: 50% effi-
However, these organic acids also have certain similarities and cacy; 3+: 75% efficacy; 4+: 95%–100% efficacy

(a) (b)

Figure 6.7 Forty-three-year-old woman with chronic eczema on both arms, before (a) and after (b) N-Ac-L-Val-L-Ala-NH2 0.5% in oil-in-
water emulsion applied twice daily for 4 weeks.

while salicylic acid diminishes the biosynthesis of dermal com- that an entire category of physiologic and nontoxic substances
ponents, i.e. glycolic acid but not salicylic acid is beneficial for can be drawn upon to repair or prevent such disfigurements.
wrinkles and aging skin. NAAs and NAPs have similar ben- The hydroxyacids and other organic acids can be utilized to
eficial effects on disturbed keratinization, e.g. ichthyosis, and nudge skin form and function toward a more youthful state.
hyperkeratoses. They can be topically applied to decelerate the otherwise inex-
N-Acetyl-L-tyrosinamide is more effective than N-acetyl- orable progression toward old-looking integument.
L-tyrosine for increasing the skin thickness by stimulation of The antioxidant PHAs and ABAs are especially benefi-
hyaluronic acid biosynthesis. N-Acetyl-L-glutamic acid diethyl cial and can be utilized to repair and prevent damage caused
ester is more effective than N-acetyl-L-glutamic acid for topical by UV radiation. However, these latter substances are still new
treatment of ichthyosis. Based on these observations, the active on the scene, and expanding use is projected. Dermatologists,
form of the NAA or NAP may not be in free acid or anion form. cosmetologists, pharmacologists, pharmacists, and formula-
Since 1974, many studies have shown that physiologic tors need to be closely familiar with chemical attributes of the
and nontoxic hydroxyacids can promote normal keratiniza- various hydroxyacids and other organic acids including PHAs
tion and increase synthesis of dermal components, including and ABAs, their clinical performance, and how best to design
hyaluronic acid and collagen fibers. AHAs, specifically glycolic and compound formulations to provide maximal achievement
acid, have been widely used in cosmetic products and derma- of intended objectives.
tologic practice for topical treatment of dry skin, acne, kerato-
ses, wrinkles, photoaging, and photodamaged skin. Because
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Retinyl Propionate and Related Retinoids

John E. Oblong

INTRODUCTION Biochemistry and Molecular Biology of

Retinoids represent a class of lipophilic compounds that Topical Retinol and Retinyl Esters
are comprised of natural derivatives of vitamin A (retinol) Of most relevance to the cosmetic industry was the confir-
as  well as synthetic analogues. Naturally-occurring reti- mation that cosmetic forms of retinoids have the potential to
noids are sourced via dietary intake of beta-carotene as well be enzymatically converted to various retinoid metabolites
as retinyl esters, both of which are found in a broad range including tRA when topically administered (3–8). The bio-
of foodstuffs and animal by-products. Structurally, beta- chemical conversions are shown schematically in Figure 7.2a,
carotene exists essentially as a dimer form of retinol and where retinol is oxidized via small chain alcohol dehydroge-
the enzymatic conversion in the intestinal tract via retinal nases to retinal and in turn to form tRA (5,9). This process is
production is the initial sourcing of retinoids for usage by begun when free retinol associates with a specific cytoplasmic
the human body. Most eukaryotic cells have the capability of retinol-binding protein (CRBP). This family of proteins with
enzymatically converting retinol and retinyl esters to various high retinoid specificity includes CRBP as well as retinoic acid
metabolites that are critical in maintaining cellular homeo- binding protein (CRABP), of which there are two isoforms,
stasis, regulating proliferation and differentiation patterns, I and II (7). These binding proteins play a direct role in regu-
as well as embryonic development. Of the metabolites gen- lation of retinoid responses in cells by acting as chaperones
erated, the true “business end” of retinoids is retinoic acid, in enzymatic conversions, sequestration, and stabilization.
which is present in cells as various cis conformations as well The retinol-CRBP complex is a substrate for retinol dehydro-
as trans-retinoic acid (tRA). Retinoids play such a critical genase, a microsomal enzyme uniquely capable of catalyzing
role in developmental biology and the metabolic pathways the conversion of retinol to retinaldehyde. Retinaldehyde is
are tightly regulated. This includes not only enzymatic pro- then rapidly and quantitatively oxidized to retinoic acid by
cesses but transport via cellular retinoid binding proteins as retinaldehyde oxidase (5,10). However, the primary reaction
well. that occurs with CRBP-bound retinol is esterification of retinol
Topical usage of potent forms of natural and syn- via lecithin:retinol acyltransferase (LRAT) or acyl CoA:retinol
thetic retinoids such as tRA has shown a high degree of acyltransferase (ARAT) to retinyl esters, the primary storage
efficacy against acne, psoriasis, icthyosis, and actinic kera- form in lipid bilayers and of which retinyl palmitate is the
tosis. Relative to photodamaged skin, tRA has clearly been major species in human skin (11,12). Recent work has identified
established as having a robust effect (1,2) and is currently the neutral lipid synthesis enzyme acyl-CoA:diacylglycerol
marketed as a prescription drug. These retinoid effects in acyltransferase 1 (DGAT1) as a novel ARAT enzyme present
diseased skin can be ascribed on some level as being a nor- in murine skin that regulates retinol oxidation to retinoic acid
malization of altered skin conditions. However, two of the (13). Additionally, retinyl stearate has been identified as the
key negatives associated with highly potent topical retinoids major form that is present in circulating serum in mammalian
are irritation that, in some instances, does not mitigate itself systems (14). This multi-step processing of retinyl esters serves
completely even after long term chronic exposure and tera- as a point of regulation to control the level of active retinoid in
togenic side effects. the skin and may thus contribute to the lower irritation poten-
Less potent topical retinoids such as retinol and vari- tial of these derivatives.
ous retinyl esters (Figure 7.1) have been building a rich his- The molecular mechanistic route that tRA follows in
tory of usage in the cosmetic marketplace. More important skin is via its function as an agonist for binding to a family
for the cosmetic marketplace is the ability of retinoids to of nuclear receptors called the retinoic acid receptors (RAR)
positively impact changes in the appearance of photodam- and which also includes the retinoid X receptors (RXR). In
aged facial skin, particularly for fine lines, wrinkles, and each of these two families, three isoforms exist and interact to
pigmentation-related changes. Additionally, understand- form heterodimers as the functionally active form. Significant
ing the basic biochemical metabolic processes that control research has gone into understanding these various dimer
endogenous retinoid levels and synthesis has shown that combinations, including agonist binding as related to various
cosmetic-type retinoids present an attractive compromise tissues and cells. Upon binding of tRA by RAR and dimeriza-
for less serious conditions such as photodamaged skin. This tion with a member of RXR, this active complex becomes a
chapter focuses on cutaneously delivered retinyl esters. The transcriptional regulator that binds to select sequences in pro-
topics covered include an overview of the metabolism, bio- moter regions of select genes, termed retinoic acid responsive
chemistry, and molecular biology as well as human study elements, or RARE (15).
findings. The principle focus in terms of treatment effects As with most biological processes that involve highly
will be upon photodamaged skin. potent agents, the retinoid pathway has several regulatory

feedback loops that help maintain an optimal level of tRA. One of how cosmetic retinoids are capable of having subtle effects
of the most important ones is depicted in Figure 7.2b, which upon photodamaged skin. As we begin to understand deeper
shows the degradation of tRA to the less active metabolites via the molecular biology changes that are caused via tRA bind-
hydroxylation to 4-hydroxy-retinoic acid and 4-oxo-retinoic ing to RAR/RXR, novel mechanistic insights into the efficacy
acid, catalyzed by members of the CYP26 family (16). As tRA as well as the negative irritation side effects could allow for an
levels become temporarily elevated, increased hydroxylase improvement in current commercialized products.
activity via higher expression levels leads to a decrease to
steady state levels. Potential to Break the Retinoid Efficacy to
In summary, the biochemical confirmation that topical Irritation Correlation with Retinyl Esters
retinol and retinyl esters can be metabolized to the more active Over the past few decades, retinol has been used extensively in
form of tRA in skin cells has provided a richer understanding cosmetic products, with the levels of retinol ranging from barely
detectable to upwards of 0.5%. The relative dosage levels vary in
part due to intolerance among consumers of irritation side effects
that is connected in part by a sensitivity to formulation differ-
ences. One approach to reduce retinoid-induced inflammation is
Retinoid Structure to attempt to regulate skin penetration and delivery rates into
CH2OH the skin via usage of nanoparticles and sponges (17). It is unclear
retinol yet whether this provides an advantage versus merely reduc-
ing delivery loads and reducing efficacy as well. As highlighted
O above, it has been generally assumed that any efficacy from topi-
CH2O C CH3 cally delivered retinol occurs via its sequential conversion via the
retinyl acetate
intermediate retinal to tRA (8). For example, it has been shown
O that 0.4% retinol can significantly improve the appearance of
CH2O C CH2CH3 wrinkles on the surface of skin but histological changes have
retinyl propionate also been measured in the extracellular matrix components of
epidermal glycosaminoglycans and dermal collagen Type I (18).
O The authors also speculated that retinol-treated skin would be
CH2O C (CH2)14CH3
retinyl palmitate more resistant to future skin injury, including ulcer formation,
among elderly consumers. These reported effects are hallmark
responses associated with topical tRA.
Several natural and synthetic esters of retinol have been
Figure 7.1 Chemical structures of retinol and retinyl esters. evaluated for retinoid-like effects, including acetate, pro-
pionate, palmitate, ascorbate, beta-glucuronide, and retinyl

(a) RAR/RXR gene


NADPH retinal
+ dehydrogenase


NADPH retinol
NADP+ hydrolase

LRAT trans-retinyl ester

(b) active inactive/less active


RA 4-hydroxylase
trans-RA OH trans-4-hydroxyRA O

Figure 7.2 Retinoid metabolic pathways in skin.


N-formyl aspartamate. While the chemical structure of retinol irritation with the one exception of retinyl propionate at 0.18%
is conducive to being esterified via the alcohol group, the abil- levels (Figure 7.3). This supports in-house clinical findings that
ity for retinyl esters to deliver efficacy that approaches retinol retinyl propionate does not cause as much irritation as reti-
becomes limited by variables such as skin penetration, cellular nol and still is able to positively impact the appearance of fine
uptake, hydrolase active site binding, re-esterification via acyl lines and wrinkles in 12-week human facial studies (21). While
transferases, and general pharmacokinetics. As with most reti- additional data would be required around the doses tested to
noid analogue work, the objective of evaluating retinyl esters confirm, these results support the suggestion that retinyl pro-
is to be able to provide a retinoid-like response on facial skin pionate is capable of eliciting a retinoid response in human
but without the negatives of irritation associated with topical skin but with a weaker overall retinoid irritation profile.
usage. In terms of application to photodamaged skin, retinyl
Since retinyl esters are dependent upon an additional propionate has recently become of interest since it is capable
biochemical processing step before releasing a free form of of affecting human skin much like retinol but with a lower
retinol (Figure 7.2a), it is anticipated that topical retinyl esters irritation profile than other retinoids (21,22, and Figure 7.3).
would have a weaker retinoid responsiveness range when While the relative activity of retinyl propionate on photodam-
compared with retinol. A human forearm biopsy study was aged skin is weaker than retinol (23), it is clear that some of
performed that evaluated via histology the effect of tRA, reti- the effects are still significant (21,22). More recently, a retinyl
nol, and three retinyl esters (acetate, propionate, and palmitate)
for the effects upon changes in epidermal and granular layer
thickness compared to a placebo control (Table 7.1). Both retinyl
acetate and retinyl propionate were able to show a significant 85
increase in both thickness measures as compared to the placebo
control. The results with retinyl propionate are similar to what 80 0.075% retinol
has been previously reported (19). These results also highlight 0.3% retinyl acetate
that there is a relative rank ordering based on the biochemi- 75
cal processing steps amongst the retinoids tested. In compar-
ing the retinyl esters, there also appears a ranking in terms Epidermal (microns) 70
0.18% retinyl propionate
of chain length from shortest to longest, with shorter chained 0.172% retinyl acetate
esters being more potent than longer chained esters. While less 65
intuitive, the potential that shorter chained retinyl esters can 0.3% retinyl propionate
be acted upon by other esterases could partially explain their 60
enhanced retinoid response profile compared with retinyl pal-
mitate, the endogenous storage form. Since the experimental 55
data in the table was performed under semi-occlusive patch,
the ability of the retinoids to penetrate into the viable cell lay- vehicle
ers is lessened as a variable for this comparison but may not
explain the complete story for retinyl palmitate (20).
As would be suspected, this rank ordering of retinoid-
0 20 40 60 80 100 120 140 160 180
based activity (efficacy) is also indirectly proportional to the
Back Scores (Redness Grades)
level of irritation that some topical retinoids can induce on
skin (20, Oblong, unpublished data). A comparison of human
retinoid activity data from Table 7.1 with irritation date from a Figure 7.3 Correlation between human irritation measures and
human back cumulative irritation protocol using nearly identi- epidermal thickening for retinyl acetate, retinyl propionate, and
cal formulations, one can see that there is a general correlation retinol.
among the esters and retinol between retinoid activity and

Table 7.1 Changes in Epidermal and Granular Layer Thicknesses in Forearm Skin due to Retinoid Treatment
Epidermal Significance vs. Granular layer Significance vs.
thickness control and other thickness control and other
Topical treatment Code (microns) treatments* (microns) treatments*
Emulsion control A 50.75 – 3.03 –
1.0% retinyl palmitate B 52.07 ns 4.88 ns
0.172% retinyl acetate C 65.45 ABGHI 10.73 ABH
0.30% retinyl acetate D 79.00 ABCeF 12.34 ABH
0.18% retinyl propionate E 68.83 ABGHI 10.61 ABH
0.30% retinyl propionate F 64.87 ABGHI 10.73 ABH
0.075% retinol G 80.13 ABCEF 14.06 AB
0.15% retinol H 85.66 ABCEF 17.44 ABCDEF
0.025% t-retinoic acid I 80.64 ABCEF 14.72 AB

Upper case letters denote significant differences ( p < 0.05), while lower case letters denote directional differences ( p < 0.10); ns = not


propionate formulation that also contained niacinamide and combat photoaged skin appearance. While the overall efficacy
collagen fragment peptides showed an equivalent level of provided by these cosmetic forms of retinoid are generally less
efficacy compared with 0.02% tRA (24). This combination of potent than the prescription forms such as tRA, the knowl-
significant efficacy with an overall lower irritation profile has edge that these forms can be metabolized to more active forms
been noted in cross-comparison of human biopsy studies, back supports the indirect mechanism of action that they utilize.
cumulative irritation, and human facial skin clinicals (21). Of the retinyl esters currently practiced in the marketplace,
While less critical than its overall efficacy and irritation profile, there appear to be advantages for the usage of retinyl propio-
retinyl propionate has been reported to have a better chemical nate over retinyl acetate in terms of having a similar efficacy
stability profile compared to other esters, thereby increasing response range but with an improved irritation profile. In con-
half-life upon skin during topical delivery (25). trast, retinyl palmitate has a very low efficacy response range,
The more potent ester retinyl acetate has been shown in rendering its low irritation profile as moot in terms of being
in vivo models as being able to induce a retinoid-like response able to have any positive benefits for affecting the appearance
as measured by epidermal thickening as well as indirect mark- photodamaged skin.
ers of epidermal proliferation (26). Additionally, the kinetics of Future research in better understanding the connections
the accommodation response to retinyl acetate as measured by between efficacy and retinoid-induced irritation should allow
erythema has a similar time curve over a 20-day period as has for the identification of novel retinyl ester that decouple these
been observed in human studies for accommodation to retinol two phenomena. The results presented with retinyl propionate
(Oblong, unpublished results). Since retinyl acetate has a similar suggest the potential exists. Additionally, there would appear
pharmaco activity and irritation profile as retinol, there appears to be an opportunity to identify a final solution that combines
to be no advantage of this ester over retinol in cosmetic products. optimized levels, formulations, and potentially additional
Of the retinyl esters currently practiced in the cos- materials to maintain or elevate retinoid-like efficacy but with
metic marketplace, retinyl palmitate appears to be one of the a reduced overall irritation profile.
weaker retinoids in terms of efficacy for generating a retinoid
response in human skin, including effects on photodamaged
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Idebenone (Hydroxydecyl Ubiquinone)

Birgit A. Neudecker, Falko Diedrich, and Howard I. Maibach

INTRODUCTION protective properties in organ preservation solutions (2). Until

Idebenone is a unique new antioxidant in dermatology used to 2009 over 270 articles have been published, mostly related
treat the visible and cellular signs of skin aging. This chapter to the health benefits of idebenone as an antiaging modality
reviews the description, uses, functionality, cell biological and (27–32). The potential benefits of idebenone fall into five catego-
clinical research, safety, and new improvements of this respi- ries: antiaging, energy enhancement, cognition enhancement,
ratory chain analog of coenzyme Q10. organ protection, and protection against excitatory amino acid
neurotoxicity. Its introduction in the field of dermatology in
2003 as a compound purported to be beneficial in the treat-
DESCRIPTION ment of many skin changes associated with acute and chronic
Idebenone is a bioengineered analog, but not a derivative
skin aging.
of ubiquinone (coenzyme Q10). It belongs in the family of
As with other antioxidants, idebenone exists in a reduced
molecules known as quinones; in particular it is a 1,4-benzo-
and an oxidized state. In a study of its effect on astroglial cells,
quinone molecule, as is hydroquinone. Its molecular weight is
idebenone, in either redox state, significantly inhibited the
338, compared with coenzyme Q10 with a molecular weight
enzymatic metabolism of arachidonic acid by cyclooxygenase
of 860, and therefore in theory has much greater skin pene-
and lipoxygenase. This effect was stronger with the reduced
tration potential. Idebenone is a potent antioxidant, with the
form, and showed potential central nervous system anti-
ability to operate under low oxygen tension situations, and not
inflammatory activity (27).
showing prooxidative metabolites (1,2). Because of its ability to
In another study, synaptosomes isolated from rat brain
inhibit lipid peroxidation, idebenone protects cell membranes
cortex were treated with iron and ascorbate, establishing
and mitochondria from oxidative damage caused by free radi-
experimental cellular oxidant injury. Idebenone prevented
cals generated during cell metabolism, processes which are
both the formation of ROS in the cytosol and mitochondria, as
also summarized as intrinsic aging (2–4). Ultraviolet radiation
well as a decrease in proteinsulfhydryl content (an indicator of
(UVR)-generated reactive oxygen species (ROS) play a critical
protein oxidation), compared with controls (28).
role in the process of photocarcinogenesis and environmen-
In addition to its function as an antioxidant, idebenone
tally induced aging, also termed as photoaging or extrinsic
works as an electron carrier in the electron transfer chain, sim-
aging (5–9). Some studies claim that infrared radiation and
ilar to coenzyme Q10. Idebenone was introduced into a canine
visible light also contribute to a major extend to such kind of
coenzyme Q10–depleted brain mitochondrial preparation,
damage (10,11). Although the skin possesses a complex anti-
which prevented the loss of electron chain transfer activity
oxidant network, both intrinsic and extrinsic pathways can
normally seen with coenzyme Q10 depletion (3).
provoke an imbalance of the fragile prooxidant-antioxidant
Idebenone also inhibited mitochondrial lipid per-
equilibrium (12–17) eventually leading to alteration of cells
oxidation (4), which can be interpreted as protecting against
and structural macromolecules of the dermal connective tissue
mitochondrial damage. Other animal studies confirm the mito-
with its clinical appearance of wrinkle formation, laxity, and
chondrial membrane protective effects of idebenone (29,30).
pigment disorders. In comparison with other antioxidants, ide-
benone shows in vitro studies as well as in vivo studies unique
properties providing protection regarding both intrinsic and
extrinsic processes of aging (18–20). FUNCTIONALITY
According to the mitochondrial theory of aging, nonrepaired
damage of mitochondrial DNA and unstable electron transfer
HISTORY AND USES cause an important loss of mitochondrial function in correlation
Idebenone has a history in medical applications dating back to with progression of age (6). Mitochondria are the sites of cellular
1982, with its initial introduction to the scientific community in metabolic energy production where oxygen is used to convert
papers published by Shimamoto et al. (21–23) citing its poten- carbohydrates into ATP to power and enable all cellular meta-
tial applications in the energy metabolism of red blood cells bolic activity. The process itself produces toxic free radicals as the
and myocardial tissue. Additional research over the next few result of electron transfer, which can result in cellular damage
years lead to the first phase I study to determine the tolerance, if uncontrolled (33,34). Therefore cells have developed defense
safety, and pharmacokinetics of idebenone (24) followed by mechanisms, including essential enzymes and coenzymes such
clinical studies in the early 1990s in subjects with Alzheimer's as coenzyme Q10, SOD, and catalase that are capable of reduc-
disease (25,26). It has since been researched extensively for a ing radical by-products of cellular energy production eventually
variety of applications related to the treatment of various age- into water (12,35). Unfortunately, these defense mechanisms are
related disorders of the human body, including cerebrovascu- efficient only to a certain degree, and liberated free radicals such
lar and Alzheimer's disease, Friedreich's ataxia, and showed as superoxide radicals can react with cellular organelles and

molecules as, for example, DNA, lipids, sugars, and proteins to 2. Protect against low-density lipoprotein (LDL) oxidation as
cause cellular damage, which expresses itself as aging (6,8,9,12). a marker of lipid peroxidation,
Mitochondria possess the ability to replicate containing their 3. Protect against cell membrane oxidation,
own DNA but have far fewer repair mechanisms than nuclear 4. Protect against DNA cross-linking post UV exposure, and
DNA despite the fact that they are the sites where most inter- 5. Protect human skin from SBC formation post UV exposure.
nal free radicals are formed. The result is cellular aging directly
associated with metabolic energy production (33). For this rea- In a scoring system, which was based on a maximum
son, mitochondria require potent free radical scavenging anti- score of 100, idebenone was found to be the most efficient anti-
oxidants to prevent the early onset of cellular aging. Coenzyme oxidant tested at the time. Since that time idebenone has been
Q10 protects mitochondria from oxidant decay. As mentioned researched in further clinical studies measuring the effects
above, the function of idebenone as an electron transfer respira- at various concentrations to reduce the overall appearance
tory chain antioxidant is closely related to that of coenzyme Q10, of fine lines, wrinkles, hyperpigmentation, and other skin
which itself is a vital biochemical found in cellular and mitochon- changes associated with photo-damage and global skin aging.
drial membranes and plays a critical role in the electron trans- The results of these studies (unpublished) are outlined below.
port chain during the production of energy in the mitochondria. Forty subjects, 6 weeks, b.i.d., double blind, global assess-
However, unlike coenzyme Q10, idebenone lacks the potential ment of 0.5% (20 subjects) and 1.0% (20 subjects) idebenone in
downside of a degradative prooxidative effect, especially under the treatment of photodamaged skin with measurements for
hypoxic cellular conditions, such as those found after stroke, skin roughness/dryness, fine lines/wrinkles, skin hydration
heart attack, excessive exercise, or other conditions that lead to and global assessment by expert dermatologist grader. The
poor tissue oxygenation. Idebenone appears to be able to tightly results are given in Figure 8.1. In this study, punch biopsies of
couple oxygen to the electron transport chain and thus prevent several patients were taken and stained for collagen 1, MMP- 1,
toxic oxygen radical production far better than coenzyme Q10 (3). IL-1, and IL-6. The results demonstrated that idebenone was
able to suppress formation of MMP, IL-1, and IL-6, and increase
deposition of collagen.
DERMATOLOGY: CELL BIOLOGICAL Low concentrations of idebenone have been shown to
AND CLINICAL RESEARCH upregulate certain important genes to suppress MMPs and inter-
It is important not to confuse idebenone with other commonly leukins and increase expression of certain structural proteins
used antioxidants in skincare such as vitamin antioxidants like for collagen (personal communication with Dr D.  McDaniel).
vitamin C and vitamin E or botanical antioxidants such as poly- Additionally, lifespan extension studies using Drosophila
phenols, flavonoids, and proanthocynidins. Idebenone differs have demonstrated the ability of idebenone to extend the lifes-
from these other types of antioxidants in that it is a respira- pan of fruit flies under oxidative stress conditions (36). Similar
tory chain antioxidant that targets aging at the mitochondria. results were found in knockout mouse models of Friedreich's
In 2003 and 2005, the first cell biological in vitro and in vivo ataxia (37).
clinical studies were published (18–20) that demonstrated that
idebenone had a potent ability to protect cellular lipids, cell
membranes, and DNA from oxidative stress and prevent sun- SAFETY
burn cell (SBC) formation post ultraviolet (UV) exposure, and As previously reviewed, sufficient preclinical and clinical data
thus could have a significant protective and corrective effect on is available to demonstrate both cell biological and clinical
skin aging. In a multistep protocol, a series of five different cell antiaging benefits of idebenone. Consultations with several
biological in vitro and clinical in vivo methods were combined companies that market idebenone in topical skincare formula-
to compare the antioxidative capacities of the following anti- tions also indicate that premarket safety HIRPT studies did not
oxidants: vitamin C, vitamin E, a-lipoic acid, coenzyme Q10, indicate any skin sensitivity concerns. However, post-market
kinetin, and idebenone. The studies included measurement of surveillance (various formulations have been on the market
the ability of the antioxidant to since 2004, including Prevage MD, Prevage, and PRIORI) sug-
gests that there have been a few reported cases of skin sensitiv-
1. Scavenge free radicals produced in a reaction chamber ity to the molecule, in most cases immediate and cumulative
using instrumental analysis (Photochem1), irritation reactions that can include skin redness, itching, and

37 37
40 33
29 30
30 26


Roughness/Dryness Fine Lines/Wrinkles Global Improvement Skin Hydration
n = 40 0.5% Idebenone 1% Idebenone

Figure 8.1 Percent increase/decrease after 6-week use.


folliculitis, but in more severe cases, what appear to be clas- damage in organ preservation solutions. Transplantation 1995;
sic allergic type reactions (38–40). Since the use of idebenone 15:60(5):444–51.
is new in skincare so it is in the general population, consum- 3. Imada I, Sato EF, Kira Y, et al. Effect of CoQ homologues on
ers reporting allergic-type reactions on first use can only be reactive oxygen generation by mitochondria. Biofactors 2008;
explained via a cross-sensitivity type model. Because the com-
4. Imada I, Fujita T, Sugiyama Y, et al. Effects of idebenone and
pound is a 1,4-benzoquinone, consumers who exhibit allergy related compounds on respiratory activities of brain mito-
to para-phenylenediamine, hydroquinone, evening primrose chondria, and on lipid peroxidation of their membranes. Arch
oil, and other similar compounds may exhibit the same sen- Gerontol Geriatr 1989; 8(3):323–41.
sitivity to idebenone. For this reason, products should carry 5. Berneburg MH, Plettenberg J, Krutmann J. Photoaging of
a caution to “patch test” products with idebenone and to wait human skin. Photodermatol Photoimmunol Photomed 2000;
at least 24 hours prior to commencing regular use. If sensitiv- 16:239–44.
ity occurs, consumers should not use products containing ide- 6. Scharfetter-Kochanek K, Brenneisen P, Wenk J, et al. Photoaging
benone. It is worthy of note here that there has never been a of the skin from phenotype to mechanisms. Exp Gerontol 2000;
recorded allergic reaction to the internal consumption of ide- 35:307–16.
7. Berneburg M, Grether-Beck S, Kurten V, et al. Singlet oxy-
benone in spite of its wide availability as a dietary supplement
gen mediates the UV-induced generation of the photoaging
in many markets around the world. associated mitochondrial common deletion. J Biol Chem 1999;
NEW IDEBENONE DERIVATIVE MOLECULES 8. Gilchrest BA. A review of skin aging and its medical therapy.
Improvements in the topical skincare delivery of idebenone Br J Dermatol 1996; 135(6):867–75.
include the development of new synthetic molecular deriva- 9. Hadshiew IM, Eller MS, Gilchrest BA. Skin aging and photoag-
ing: The role of DNA damage and repair. Am J Contact Dermatol
tives, which seem to have the capability to enhance efficacy and
2000; 11:19–25.
safety of this new antioxidant technology. Specifically, various 10. Schieke SM, Schroeder P, Krutmann J. Cutaneous effects of
water and oil soluble esters of idebenone were synthesized infrared radiation: From clinical observations to molecular
and tested for their ability to inhibit SBC formation post- response mechanisms. Photodermatol Photoimmunol Photomed
UV irradiation, and the first clinical studies were conducted 2003; 19(5):228–34.
with assessments for known antiaging parameters including 11. Cho S, Shin MH, Kim YK, et al. Effects of infrared radiation and
red and brown pigmentation, fine lines and wrinkles, and heat on human skin aging in vivo. J Investig Dermatol Symp Proc
global improvement in photodamage. Finally, skin maximiza- 2009; 14(1):15–19.
tion studies were conducted to asses skin tolerance. In these 12. Thiele JJ, Schroeter C, Hsieh SN, et al. The antioxidant network
pilot studies the dipalmitic glyceric acid ester of idebenone of the stratum corneum. Curr Probl Dermatol 2001; 29:26–42.
13. Podda M, Traber MG, Weber C, et al. UV-irradiation depletes
(hydroxydecyl ubiquinoyl dipalmitoyl glycerate) was found to
antioxidants and causes oxidative damage in a model of human
be the most efficient new idebenone derivative molecule across skin. Free Radic Biol Med 1998; 24(1):55–65.
all test parameters (unpublished study, personal communica- 14. Wenk J, Brenneisen P, Meewes C, et al. UV-induced oxidative
tion with Dr. D. McDaniel). stress and photoaging. Curr Probl Dermatol 2001; 29:83–94.
It is believed that the improved antioxidant and anti- 15. Darr D, Pinell SR. Reactive oxygen species and antioxidative
aging efficiency of the new derivative is a result of increased protection in photodermatology. In: Lowe NJ, Shaath NA,
skin and cell permeability, improved time-released action of Oathak MA, eds. Sunscreens - Development, Evaluation, and
idebenone based on rate constant enzymatic hydrolysis in the Regulatory Aspects. 2nd ed. New York: Dekker 1997; pp. 155–173.
skin, leading to broader skin compatibility, and more efficient 16. Fuchs J, Packer L. Oxidative Stress in Dermatology. New York,
antioxidant capacity. Basel: Dekker 1993; pp. 55–73.
17. Sander CS, Chang H, Salzmann S, et al. Photoaging is asso-
ciated with protein oxidation in human skin in vivo. J Invest
CONCLUSION Dermatol 2002; 118(4):618–25.
Idebenone is a very promising new unique respiratory chain 18. Neudecker BA, Bekyarov D, Diedrich FK, et al. Antioxidants
antioxidant for topical skincare use in treating various signs compared in a multiphasic protocol to measure to measure pro-
of skin aging. In cell biological studies it has been shown to tective capacity against oxidative stress. Poster presentation,
protect cellular lipoproteins, cell membranes, and DNA from American Academy of Dermatology, San Francisco, 2003.
19. McDaniel DH, Neudecker BA, Dinardo JC, et al. Idebenone: A
damage caused by oxidative stress. Clinically it has an anti-
new antioxidant - part I. Relative assessment of oxidative stress
inflammatory effect, the ability to reduce SBCs post UV expo- protection capacity compared to commonly known antioxi-
sure, and the ability to diminish the visible signs of skin aging dants. J Cosmet Dermatol 2005; 4(1):10–17.
including fine lines, wrinkles, hyperpigmentation, and over- 20. McDaniel D, Neudecker B, Dinardo J, et al. Clinical efficacy
all improvement in photodamaged skin. Improvements in the assessment in photodamaged skin of 0.5% and 1.0% idebenone.
technology have produced new idebenone derivative mole- J Cosmet Dermatol 2005; 4(3):167–73.
cules; in particular, dipalmitic glyceric acid, which seems to 21. Shimamoto N, Goto N, Hirata M. Effects of 2,3-dimethoxy-
improve the delivery of the molecule and its efficacy but also 5-methyl-6-(100-hydroxydecyl)-1,4-benzoquinone (CV-2619)
significantly decreases the potential for skin sensitivity. on the energy metabolism of red blood cells of rats. Nippon
Yakurigaku Zasshi 1982; 80(2):137–45.
22. Shimamoto N, Tanabe M, Imamoto T, et al. Effects of 2,3-
REFERENCES dimethoxy-5-methyl-6-(100-hydroxydecyl)-1,4-benzoquinone
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properties of 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)- trophied heart of spontaneously hypertensive rats. Nippon
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11(1):54–63. 23. Shimamoto N, Tanabe M, Hirata M. Effects of 2,3-dimethoxy-
2. Wieland E, Schutz E, Armstrong VW, et al. Idebenone pro- 5-methyl-6-(100 -hydroxydecyl)-1,4-benzoquinone (CV-2619)
tects hepatic microsomes against oxygen radical-mediated on adriamycin-induced ECG abnormalities and myocardial

energy metabolism in spontaneously hypertensive rats. Nippon metabolites in the brains of rats with cerebral ischemia. Arch
Yakurigaku Zasshi 1982; 80(4):307–15. Gerontol Geriatr 1989; 8(3):247–56.
24. Barkworth MF, Dyde CJ, Johnson KI, et al. An early phase I study 32. Kakihana M, Yamazaki N, Nagaoka A. Effects of idebenone
to determine the tolerance, safety and pharmacokinetics of (CV- 2619) on the concentrations of acetylcholine and choline
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1985; 35(11):1704–7. Pharmacol 1984; 36(3):357–63.
25. Marigliano V, Abate G, Barbagallo-Sangiorgi G, et al. 33. Krutmann J, Schroeder P. Role of mitochondria in photoag-
Randomized, double-blind, placebo controlled, multicentre ing of human skin: The defective powerhouse model. J Investig
study of idebenone in patients suffering from multi-infarct Dermatol Symp Proc 2009; 14(1):44–9.
dementia. Arch Gerontol Geriatr 1992; 15(3):239–48. 34. Cardoso SM, Pereira C, Oliveira R. Mitochondrial function is
26. Senin U, Parnetti L, Barbagallo-Sangiorgi G, et al. Idebenone in differentially affected upon oxidative stress. Free Radic Biol Med
senile dementia of Alzheimer type: A multicentre study. Arch 1999; 26(1–2):3–13.
Gerontol Geriatr 1992; 15(3):249–60. 35. Shindo Y, Witte E, Han D, et al. Enzymatic and nonenzymatic
27. Civenni G, Bezzi P, Trotti D, et al. Inhibitory effect of the neuro- antioxidants in epidermis and dermis of human skin. J Invest
protective agent idebenone on arachidonic acid metabolism in Dermatol 1994; 102:122–4.
astrocytes. Eur J Pharm 1999; 370:161–7. 36. Seznec H, Simon D, Monassier L, et al. Idebenone delays the
28. Cardoso SM, Pereira C, Oliveira CR. The protective effect of onset of cardiac functional alteration without correction of Fe-S
vitamin E, idebenone and reduced glutathione on free radical enzymes deficit in a mouse model for Friedreich ataxia. Hum
mediated injury in rat brain synaptosomes. Biochem Biophys Res Mol Genet 2004; 13(10):1017–24.
Commun 1998; 246(3):703–10. 37. Puccio H. Multicellular models of Friedreich ataxia. J Neurol
29. Nagaoka A, Kakihana M, Fujiwara K. Effects of idebenone 2009; 256(suppl 1):18–24.
on neurological deficits following cerebrovascular lesions in 38. McAleer MA, Collins P. Allergic contact dermatitis to
stroke-prone spontaneously hypertensive rats. Arch Gerontol hydroxydecyl ubiquinone (idebenone) following application of
Geriatr 1989; 8(3):203–12. antiageing cosmetic cream. Contact Dermatitis 2008; 59(3):178–9.
30. Nagaoka A, Suno M, Shibota M, et al. Effects of idebenone 39. Natkunarajah J, Ostlere L. Allergic contact dermatitis to ide-
on neurological deficits, local cerebral blood flow, and energy benone in an over-the-counter anti-ageing cream. Contact
metabolism in rats with experimental cerebral ischemia. Arch Dermatitis 2008; 58(4):239.
Gerontol Geriatr 1989; 8(3):193–202. 40. Sasseville D, Moreau L, Al-Sowaidi M. Allergic contact der-
31. Kakihana M, Yamazaki N, Nagaoka A. Effects of idebenone on matitis to idebenone used as an antioxidant in an anti-wrinkle
the levels of acetylcholine, choline, free fatty acids, and energy cream. Contact Dermatitis 2007; 56(2):117–8.

Frank Dreher

INTRODUCTION ⦁O − gives hydrogen peroxide (H O ) by a dismutation reaction

2 2 2
The skin forms an efficient barrier limiting chemicals from either spontaneously or catalyzed by cutaneous superoxide
entering our body and protects from the harmful environment dismutase (SOD). Further, in the presence of metal ions such
encompassing exposure to solar ultraviolet radiation (UVR) as Fe(II) or Cu(II), H2O2 can be converted to the highly reac-
and air pollutants. Such an exposure results in the formation tive hydroxyl radical ⦁OH. Otherwise (major type II reaction),
of reactive oxygen (ROS) and reactive nitrogen species (RNS). electronically excited and reactive singlet oxygen 1O2 is formed
As oxidative stress-promoting chemicals, those species can by photoenergy transfer from UVR-excited chromophores in
react with skin biomolecules what may ultimately lead to skin the presence of triplet oxygen 3O2 (molecular oxygen in its
damage (1,2). To counteract ROS- and RNS-induced oxida- ground state). Following their formation, ROS species includ-
tive stress, the skin is equipped with a variety of antioxidants ing 1O2, ⦁O2−, ⦁OH, and H2O2 react with an array of skin bio-
forming an antioxidant network. The antioxidants intervene molecules including lipids, proteins, DNA, and carbohydrates.
at different levels of oxidative processes by scavenging and For instance, (poly)unsaturated lipids (LH) may react with
removing free radicals or oxidatively damaged biomolecules. ROS forming lipid peroxyl (LOO⦁) and alkoxyl radicals (LO⦁),
However, the antioxidant defense in skin can be overwhelmed which may initiate a chain-propagating autocatalytic reaction.
by an increased exposure to oxidative stress, or may be com- ROS can also cause modifications of amino acids of proteins
promised due to malnutrition or certain health conditions. resulting in functional changes of structural or enzymatic pro-
Well documented solar UVR-induced skin damage teins. Besides a multitude of ROS-mediated DNA damages,
includes acute reactions including sunburn. Premature skin reaction of singlet oxygen with DNA results in the formation
aging (photoaging) and photocarcinogenesis are the conse- of 8-hydroxy-deoxyguanosine. Since DNA absorbs strongly in
quences of chronic UVR exposure. Terrestrial solar UVR con- the UVB region and is only a weak chromophore in the UVA
sists of UVB (290–320 nm) and UVA (UVA II: 320–340 nm, UVA region, UVB is largely considered as a direct, ROS-independent
I: 340–400 nm). Radiation less than 290 nm (UVC) does not inducer of DNA damage. UVB absorption of DNA leads to
reach the earth's surface, since these wavelengths are absorbed major base modifications such as pyrimidine dimer or (6–4)
by stratosperic ozone. While ozone in the upper atmosphere photo-adduct formation. These modifications together with
occurs naturally and protects skin by filtering out harmful indirect DNA damage induced by ROS are involved in solar
solar UVR, ozone at ground level (troposphere) is a noxious, genotoxicity.
highly reactive oxidant pollutant. Besides UVR and air pollut- Additionally, oxidative damage to mitochondrial DNA
ants such as tropospheric ozone, the presence of chemically (mtDNA) has been described to play a major role in photoag-
unstable and ROS/RNS-forming drugs as well as the exposure ing (3). Studies have shown that a common deletion of mtDNA
of skin to photoreactive chemicals may be other sources of is increased about 10-fold in photoaged skin as compared to
cutaneous oxidative stress. Recently, it was found that also the sun-protected skin in the same individual (4). Although nor-
visible light and infrared parts of the sun spectrum appear to mal ATP production in the mitochondria results in some level
contribute to oxidative stress in skin. of oxidative stress, it is thought that UVA exposure increases
oxidative stress in the mitochondria what leads to mutations
of mtDNA and consequently to a defective respiratory chain.
REACTIVE OXYGEN SPECIES The defective respiratory chain results in reduced energy pro-
Several steps lead to the formation of ROS during UVR expo- duction by the mitochondria and increased production of ROS
sure, which represents a well characterized source of oxidative (5–8). This increase in ROS leads to more mtDNA mutations
stress in skin (1,2). The cascade of ROS formation is initiated by which further perpetuate the production of ROS. As a conse-
UVA absorption of endogenous or exogenous chromophores quence, mtDNA mutations will increase even in the absence of
present in the skin. Of the many skin constituents capable of UV exposure (3,9). This hypothesis has been termed “the defec-
absorbing UVA, trans-urocanic acid, melanins, flavins, por- tive powerhouse model of premature skin aging” (3).
phyrins, protein-bound tryptophan, or advanced glycation
end products are believed to be relevant chromophores initi-
ating the ROS formation cascade. Following UVR absorption, CONSTITUTIVE SKIN ANTIOXIDANT NETWORK
the activated chromophore may react in two ways. In type I To protect against oxidative stress, skin is equipped with a
photoreactions, the excited chromophore directly reacts with a complex network of enzymatic and nonenzymatic antioxi-
substrate molecule via electron or hydrogen atom transfer and dants (1,2). Antioxidant enzymes such as SOD, catalase, glu-
gives rise to free radical formation. In the presence of molecu- tathione reductase and peroxidase, glutathion-S-transferase
lar oxygen (minor type II reaction), this reaction may lead to and thioredoxin reductase, and peroxidase interact with low
the formation of superoxide anion radical ⦁O2− Subsequently, molecular weight lipophilic antioxidants including vitamin E

homologues (tocopherols and tocotrienols) and ubiquinols to dehydroascorbic acid and then irreversibly decomposed.
(coenzyme Q) as well as hydrophilic antioxidants such as Glutathione also reacts with singlet oxygen, superoxide
vitamin C (ascorbic acid or ascorbate) and glutathione (GSH) anion radicals, and hydroxyl radicals resulting in the forma-
(Figure 9.1). Carotenoids, retinoids, and uric acid, also possess- tion of the thiyl radical GS ⦁ and subsequently glutathione
ing antioxidant activity, were also found in skin. Their role disulfide GSSG. The latter can be recycled to GSH by the
within the cutaneous antioxidant network is, however, less NAD(P)H-dependent enzyme glutathione reductase.
well understood. GSH is also a cofactor for a few reducing enzymes,
α-Tocopherol, the predominant vitamin E homologue among them glutathione peroxidases. Glutathion peroxidase
in skin, is known to efficiently scavenge lipid peroxyl and alk- utilizes lipid peroxides as substrate and converts them into
oxyl radicals by intercepting lipid chain propagation, which hydroxy fatty acids. Glutathion peroxidase also catalyzes the
results in the formation of the metastable tocopheroxyl radi- conversion of H2O2 into water and oxygen. As mentioned,
cal. This radical then either reacts with another lipid radical, less reactive H2O2 is produced by SOD catalyzing the dismu-
leading to α-tocopherol consumption, or abstracts a hydro- tation reaction of superoxide anion radicals. SOD is present
gen atom from polyunsaturated lipids to give α-tocopherol in skin as Cu/Zn- and Mn-SOD. GSH is likewise used by
and lipid radical. In the latter case, occurring preferentially glutathion-S-transferases, which catalyze the conjugation of
at low lipid radical concentration, the lipid radical may later GSH to a variety of electrophils, including oxidized lipids,
react with oxygen to form a lipid peroxyl radical. This reac- DNA, and other molecules. Glutathion-S-transferases there-
tion consequently induces the α-tocopherol–mediated lipid fore play an important role in removing products of oxida-
peroxidation chain reaction. Formation of one molecule of tive stress.
α-tocopherol radical results in the formation of many lipid Skin also contains catalase, which is similar to gluta-
hydroperoxides. However, as demonstrated in vitro in lipid thione peroxidase and eliminates H2O2. However, catalase
and cellular systems, when ascorbic acid or ubiquinol are contributes to scavenging H2O2 differently than glutathione
present, the tocopheroxyl radical is rapidly reduced, regen- peroxidase with respect to its cellular distribution, enzyme
erating α-tocopherol. The α-tocopherol–mediated lipid per- stability, and reaction rate. The enzymatic activity of cata-
oxidation chain reaction is thereby terminated. In addition, lase is much higher than that of glutathione peroxidase in
because of its high reduction potential, ascorbic acid is an human epidermis (10). Besides GSH peroxidase, skin contains
efficient scavenger of a series of ROS such as superoxide another selenium-dependent enzyme, thioredoxin reductase
anion radicals, hydroxyl radicals, singlet oxygen, as well as (11). Thioredoxin reductase, together with its electron accep-
water-soluble peroxyl radicals. The resulting ascorbic acid tor thioredoxin and thioredoxin peroxidase, participates in the
radical can be either recycled to ascorbic acid by co-antioxi- cutaneous H2O2 turnover similarly to the enzymic thiol redox
dants such as glutathione or, respectively, is further oxidized couple GSH reductase/peroxidase.

GSH or
α-Tocopheroxyl Glutathione
radical disulfide,
Thioredoxin (ox), NAD(P)+
α-Lipoic acid

Vitamin E
interface Thiol
Ascorbic Cycle
LOOH, acid
α-Tocopherol Glutathione,
Lipids Vitamin C Thioredoxin (red),
(LH ) Cycle Dihydrolipoic acid,

Singlet oxygen,
Hydroxyl radical,
Superoxide radical
Superoxide radical
dismutase Oxidative stressors
(UVA, UVB, ozone, etc.)
H2O + Catalase
1/2O H2O2
2 GSH-peroxidase

Figure 9.1 Postulated activation of interactive network of antioxidants and antioxidant enzymes by oxidative stress in skin; note that
some of the depicted antioxidant recycling mechanisms have been found in vitro and in other than cutaneous systems.

Along with skin's “interceptive” antioxidant network EFFECTS OF ENVIRONMENTAL

that scavenges ROS and RNS, skin also possesses a mecha- STRESSORS ON SKIN ANTIOXIDANTS
nism of “antioxidant repair” that is able to reverse oxidatively Numerous studies have documented the effects of UVR on
damaged proteins (12). In general, nonenzymic antioxidant cutaneous antioxidants after acute or chronic exposure using
concentrations as well as enzymic antioxidant activities are different animal models, but fewer studies exist that inves-
significantly higher in the epidermis as compared to the tigate the mechanisms and consequences of such effects in
dermis. This probably reflects the fact that the epidermis is humans (1,2). The antioxidants contained in the stratum cor-
directly exposed to various exogenous sources of oxidative neum have been demonstrated to be susceptible to UVR. For
stress and might have therefore evolved to possess a more example, a single suberythemal dose of solar-simulated UVR
pronounced antioxidant defense capacity than the dermis. depleted human stratum corneum α-tocopherol by almost half,
On a molar basis, hydrophilic nonenzymatic antioxidants while dermal and epidermal α-tocopherol were only depleted
including L-ascorbic acid, GSH, and uric acid appear to be at much higher doses (14). The high susceptibility of stratum
the predominant antioxidants in human skin. Their dermal corneum vitamin E to UVR may be, at least in part, due to a lack
and epidermal overall concentrations are more than approxi- of co-antioxidants in the outermost skin layer. The lipophilic
mately 10- to 100-fold greater than found for vitamin E or antioxidant ubiquinone-10 (oxidized form of ubiquinol-10), the
ubiquinol/ubiquinone. Ascorbic acid, GSH, uric acid (13), most abundant ubiquinol/ubiquinone found in human skin,
and vitamin E (14) as well as catalase and SOD (15,16) were was undetectable in human stratum corneum.
also detected in the outermost epidermal layer, the human Additionally, ascorbic acid, the major hydrophilic
stratum corneum. On the other hand, glutathione peroxi- co-antioxidant that is also capable of recycling photooxidized
dase activity seems not to be detectable in human stratum α-tocopherol, is present at lower levels in human stratum cor-
corneum (16). It was found that the distribution of antioxi- neum than in other skin tissues. Because stratum corneum
dants in stratum corneum is not homogeneous, but follows a represents a compartmentalized structure, the antioxidants
gradient with lower concentrations toward the skin surface are probably not homogeneously distributed. This may further
(14). Such a gradient may be explained by the fact that the affect their interactions and thus limit the recycling capacity of
outer skin layers are more directly exposed to environmental α-tocopherol. The hydrophilic antioxidants were also shown to
sources of ROS. A second reason may be related to the longer be sensitive to UVR. Direct depletion of α-tocopherol and for-
exposure of the superficial stratum corneum layers to oxida- mation of its radical may further affect these endogenous anti-
tive stress as compared to the lower layers, as a consequence oxidant pools. However, it seems that ascorbic and uric acid are
of the physiological turnover of keratinocytes during their less susceptible to solar-simulated UVR than α-tocopherol or
differentiation process. Interestingly, while human stratum ubiquinol-10, as shown with cultured human skin models (23).
corneum concentrations of vitamin E are as high as in lower In full thickness epidermis of hairless mice, however, ascor-
epidermal layers, concentrations of hydrophilic antioxidants bic acid was depleted at lower solar-simulated UV doses than
ascorbic acid and uric acid are in the range of one to two those needed to deplete lipophilic antioxidants or GSH (24). In
orders of magnitude lower. This is most likely due to the another study, murine epidermal GSH levels were significantly
decreased water content of the human stratum corneum as depleted within minutes after UVB exposure but returned to
compared with less keratinized epidermal layers. In contrast normal levels after half an hour (25). Moreover, exposures
to uric acid, GSH, and ubiquinol, ascorbic acid and the vita- of hairless mice to solar simulated UVR demonstrated that
min E homologues cannot be synthesized by humans and dermal and epidermal catalase is more susceptible to photo-
must be taken up by the diet. Consequently, the skin's antiox- inactivation than SOD, and far more than GSH peroxidase and
idant defense is dependent on nutritive factors. Knowledge GSSG reductase (26,27).
of the physiological regulation of ascorbic acid and vitamin E Effects of the air pollutant ozone on skin antioxidants
in skin is emerging. For instance, once ascorbic acid reaches have also been reported (1,2). Similarly, as found for UVR
skin via dermal blood vessels, it eventually enters the dermis exposure, the stratum corneum is the most susceptible skin
where it is taken up by fibroblasts using a specific, sodium- layer for ozone-induced depletion of lipophilic and hydro-
dependent vitamin C transporter (SVCT) 2, or further dif- philic antioxidants, as was demonstrated using hairless mice.
fuses through the dermis, finally reaching the epidermis and Ozone itself is too reactive to penetrate deeply into the skin and
supplying keratinocytes mainly via SVCT1 (17). reacts therefore predominantly with the skin barrier lipids and
α-Tocopherol is known to be a significant constituent of proteins in the outermost epidermis. Based on transepidermal
human sebum and is continuously secreted to the skin surface water loss changes measured in hairless mice after exposure to
(18). Similarly as for carotenoids (19), sebaceous gland secre- either solar-simulated UVR or repetitive high doses of ozone,
tion is believed to be a relevant physiological delivery pathway UVR appears a more damaging source of oxidative stress than
of α-tocopherol to sebaceous gland-rich skin regions, such as ozone for skin (28).
the well-exposed facial skin. This may explain the increased A complex regulation in the antioxidant system of human
level of α-tocopherol detected in the upper stratum corneum of skin was revealed during aging processes (15,29). α-Tocopherol
facial skin as compared to upper arm skin. The physiological concentrations were significantly lower in the epidermis of
role of vitamin E in human sebum may be to particularly limit aged skin but not for the dermis. Ascorbic acid levels were
the formation of toxic skin surface lipid photooxidation prod- lower in both epidermis and dermis of aged skin. Total gluta-
ucts, such as squalene mono-hydroperoxides (20). In addition thione levels were also lower, whereas uric acid concentrations
to its antioxidant activity, L-ascorbic acid acts as cofactor in a were constant in the epidermis and dermis. Moreover, protein
multitude of metabolic processes involved in skin formation. oxidation is increased in intrinsically aged, and, most signifi-
For example, it is required in hydroxylation reactions during cantly, in photoaged human skin. Here, the oxidative damage
collagen synthesis to form connective tissue (21) and partici- is most pronounced in the papillary dermis and correlates well
pates in biosynthesis of epidermal barrier lipids (22). with solar elastosis. Remarkably, both protein oxidation as well

a sharp decline in catalase protein levels were also found in and sunburn cell formation when formulated at higher concen-
the stratum corneum; however, not in the lower epidermal lay- trations (e.g., 15%) in an appropriate vehicle at low pH (e.g., pH
ers, where antioxidant protection is overall higher than in der- 3.2) (39,40). In a human study, however, a hydroalcoholic lotion
mal and stratum corneum layers (15). Accordingly, an age- and with 5% vitamin C was unable to induce any significant photo-
UVR-dependent decline of stratum corneum catalase enzyme protective effects when applied once 30 minutes before irradia-
activity was later demonstrated (16). tion at a dose of 2mg/cm2 (35). Besides differences between pig
and human skin responses, differences in vitamin C concen-
PHOTOPROTECTION OF HUMAN SKIN tration, mode of application including applied dose and vehi-
BY TOPICAL ANTIOXIDANTS cle composition, as well as other experimental parameters may
Apart from using sunscreens to diminish the intensity of UVR explain this difference in the reported photoprotective efficacy
reaching the deeper skin layers, supplementation of the skin of the vitamin C formulations.
with topically applied antioxidants and thereby strengthening The development of a stable formulation containing
its antioxidative capacity is an established approach in limit- vitamin C is challenging since vitamin C is easily oxidized.
ing oxidative stress–induced skin damage (1,2,30). Oral supple- Vitamin C can be stabilized in an aqueous formulation at
mentation of antioxidants, which is another strategy to prevent low pH when kept under oxygen exclusion (41). Newly, spe-
photodamage of skin, is not the subject of this chapter and has cial water-free, silicon-based preparations allow stabilizing
been reviewed elsewhere (31–33). Topical application of anti- vitamin C over a prolonged period of time (42). Additionally,
oxidants provides an efficient means of increasing antioxidant esterified vitamin C derivatives such as magnesium or sodium
tissue levels in human skin. As the most susceptible skin layer ascorbyl phosphate, aminopropyl ascorbyl phosphate, and tet-
for UVR- and ozone-induced depletion of cutaneous antioxi- rahexyldecyl ascorbate (tetra-isopalmitate ascorbate) are stable
dants, the stratum corneum may particularly benefit from an alternatives to vitamin C (43,44). However, as described for
enhanced antioxidant capacity after topical supplementation. vitamin E esters, these esters must be hydrolyzed to vitamin C
to reveal antioxidant properties. This will take time and may
Vitamin E occur only to a limited extent. Furthermore, some of those
The photoprotective effects of vitamin E (α-tocopherol) have derivatives (e.g., tetrahexyldecyl ascorbate) are of significantly
been studied extensively. Most studies were performed in higher molecular weight as compared to vitamin C, that can
animals, but several studies also exist investigating the pho- lower their skin permeability and consequently also their anti-
toprotective effects of topically applied vitamin E in humans oxidant efficacy after topical application.
(1,2,34). Significantly reduced acute skin responses such as ery- Vitamin C does not act as sunscreen, nor does it absorb
thema and edema, sunburn cell formation, lipid peroxidation, UVA. In addition to its antioxidant properties, vitamin C par-
DNA adduct formation, immunosuppression, as well as UVA- ticipates in the formation of collagen fibers as a cofactor of pro-
induced binding of photosensitizers was demonstrated when lyl and lysyl hydroxylase, enzymes essential for the stabilizing
vitamin E was applied before UVR exposure. As shown in ani- and cross-linking of newly synthesized collagen molecules. A
mal studies, skin wrinkling and skin tumor incidence due to human placebo-controlled study demonstrated that the use of
chronic UVR exposure seem also to be diminished by topical a 5% vitamin C cream resulted in significantly improved skin
vitamin E. A human study proved that an alcoholic lotion con- relief and a decrease in deep furrows after 6 months of use (45).
taining 2% α-tocopherol significantly diminished the erythe-
mal responses when applied 30 minutes before UVR exposure Polyphenols
(35). While this lotion had no sunscreening properties, some Compounds from dietary and medical plants and vegetables
α-tocopherol preparations may also act as sunscreens (36). have gained considerable attention as promising agents in
Diverse vitamin E esters, in particular vitamin E ace- protecting skin from UVR-induced photodamage after topical
tate, were also shown to reduce UVR-induced skin damage. application (46–49). Extracts from green tea, wine grapes, cof-
However, their photoprotective effects are less pronounced fee berry, feverfew, milk thistle, pomegranate, tropical ferns,
as compared to vitamin E. Vitamin E esters need to be hydro- and turmeric were particularly well studied. They contain a
lyzed during skin absorption to show antioxidant activity. For wide variety of polyphenols known as flavonoids, which are
instance, bioconversion of vitamin E acetate into α-tocopherol, divided into flavonols, flavones, catechins, flavanones, antho-
its active antioxidant form, seems slow and occurs only to cyanidins, and isoflavonoids. They are synthesized conjointly
some extent. There is evidence that vitamin E acetate is not with ascorbic acid, vitamin E, and GSH by plants as a response
hydrolyzed in the stratum corneum and that its bioconversion to mitigate cellular damage under oxidative conditions. Their
into α-tocopherol only occurs after penetration into the nucle- antioxidant properties arise from their high reactivity as
ated epidermis (37). Consequently, the controversial observa- hydrogen or electron donors, from the ability of the polyphe-
tions of photoprotective effects of topically applied vitamin nol-derived radical to stabilize the unpaired electron, as well
E acetate may be explained by a limited bioavailability of the as from their ability to chelate transition metal ions such as
active, ester-cleaved form during oxidative stress at the site of Fe(II), thereby interfering with hydroxyl radical production.
action. Intriguingly, the bioconversion of vitamin E acetate into Besides hydroxyl radicals, polyphenols are believed to quench
its active form can be enhanced when skin is exposed to sun, singlet oxygen, superoxide anion radicals, and peroxyl radi-
possibly by an UVB dependent increase in esterase activity as cals. Polyphenolic compounds also possess anti-inflammatory
demonstrated in murine epidermis (38). and other properties beneficial for skin.
Green tea (Camellia sinensis) extracts are the best studied
Vitamin C plant-derived antioxidants for skin (50). In contrast to black tea,
Several studies investigated the photoprotective effects of topi- which is fermented, green tea leaves contain a high concentra-
cal vitamin C (L-ascorbic acid). Using a porcine skin model, top- tions of polyphenols such as epigallocatechin gallate (EGCG).
ically applied vitamin C protects from UVB-induced erythema Green tea polyphenols act as antioxidants by scavenging ROS/

RNS, by sequestering metal ions, and act indirectly as antioxi- ester, and N-acetylcysteine, respectively, efficiently protected
dants through inhibition of “pro-oxidant” enzymes such as against UVB radiation–induced epidermal lipid peroxidation,
inducible nitric oxide synthase, lipoxygenases, and cycloxy- cytotoxicity, and apoptosis using pig skin ex vivo as a skin
genases, and further induce the antioxidant enzymes GSH-S- model (65). However, their photoprotective effects have been
transferases and SOD (51). Protective effects of green tea extract reported in only a few clinical studies. Topical treatment with
and its major polyphenolic constituent EGCG on UVR-induced N-acetylcysteine under occlusion resulted in an increased
skin damage after topical application were first observed in GSH level and eliminated its oxidized form (GSSG) in humans
animals (50). These effects were later confirmed in humans, (66). Thus, the additional stimulation of GSH biosynthesis
where topical application of green tea extracts or EGCG sig- might be a key mechanism accounting for the observed photo-
nificantly decreased erythema responses, lipid peroxidation, protective effects of N-acetylcysteine. Dihydrolipoic acid, the
and DNA damage (52,53). More recently, a placebo-controlled reduced and primarily active antioxidant form of α-lipoic acid,
study demonstrated that the combined use of a 10% green tea is another promising thiol antioxidant (67).
cream and oral green tea supplementation for 8 weeks resulted
in a significant improvement in elastic tissue (54). A trend
toward improvement but no significant differences in clinical
Other Antioxidants
The pineal hormone melatonin has antioxidant properties. It
grading were found between the green tea–treated and the
has been shown to significantly reduce UVR-induced erythema
placebo group, indicating that a longer treatment period may
in humans, although its potential sunscreening properties
be required for clinically relevant improvements. In another
as well as its immunomodulatory function may have contrib-
placebo-controlled study, topical application of a green tea pro-
uted to the observed photoprotective effects (35). In addition,
tected human skin from solar-simulated ultraviolet light when
L-ergothioneine, a thiourea derivative of histidine found in
applied 15 minutes prior to exposure and reapplied immedi-
food plants and mushrooms, is another potent antioxidant suit-
ately after exposure to two minimum erythema doses (55).
able for topical application (68). Idebenone, a synthetic analogue
A further study showed that three-time daily use of a lotion
of coenzyme Q, is another interesting agent with antioxidant
containing 0.4% of a green tea extract with 40% to 50% total
properties (69). Whereas a clinical study with a 1% idebenone
polyphenol content helped to reduce UVB-mediated increase
formulation demonstrated a reduction in wrinkles in females
in sunburn cell formation and p53 expression in keratinocytes,
with moderate photodamage (70), a study in pigs revealed that
but did not reduce erythema or formation of thymidine dimers
idebenone seems to offer little to no photoprotective effects (71).
(56). This study indicated that formulations with relatively low
concentrations of green tea extracts can be sufficient for pro-
viding photoprotection. Green tea extracts and EGCG were Antioxidant Combinations
further shown to have chemopreventive effects in rodents, and Antioxidants interact when combined and emanating radi-
limit cancer formation. However, epidemiological studies with cal or oxidized forms of antioxidants after ROS scavenging
green tea are so far not conclusive (50,57). may be quickly regenerated in the presence of appropriate
Numerous other polyphenols have also been studied. co-antioxidants. Accordingly, an enhanced photoprotective
For instance, a milk thistle extract containing silibinin as effect may be obtained by applying distinct combinations
predominant polyphenol was shown to inhibit UVB-induced of antioxidants. Ample evidence exists about the interac-
immunosuppression, reduce UVB-induced sunburn cell for- tive dependence of vitamins C and E in diminishing photo-
mation, prevent DNA adduct formation, and prevent photocar- damage. A single topical application of a combination of 2%
cinogenesis after topical application in mice (58,59). In another vitamin E and 5% vitamin C resulted in a significantly more
study, topical administration of genistein substantially inhib- pronounced photoprotective effect as compared to the appli-
ited UVR-induced hydrogen peroxide formation, lipid peroxi- cation of either antioxidant alone in the identical vehicle (35).
dation, and DNA damage in mice, and protected human skin As demonstrated in the same clinical study, the most dra-
against UVB-induced erythema (60). Furthermore, topical matic improvement resulted from the combination of mela-
application of a tropical fern extract reduced erythema when tonin with α-tocopherol and ascorbic acid. Other mixtures of
applied before UVR exposure as shown in humans (61). In a topically applied antioxidants were also shown to be more
clinical study, 1% coffee berry extract (containing diverse (poly) effective in reducing photodamage than single antioxidants.
phenolic compounds including chlorogenic acid, quinic acid, Adding 0.5% ferulic acid (a phenolic antioxidant found in
and ferulic acid) resulted in a significant improvement in signs plants) to a solution of 1% α-tocopherol and 15% ascorbic
of skin aging when compared to vehicle (62). Pomegranate acid doubled photoprotection to solar-simulated irradia-
fruit extract, comprising the polyphenol ellagic acid, possesses tion as measured by both erythema and sunburn cell forma-
strong antioxidant and anti-inflammatory properties, and lim- tion in pigs (72). Another combination consisting of ferulic
ited UVB-mediated damage in a human reconstituted skin acid with tocopheryl acetate and α-glycosylrutin limited
model (63). Another natural extract, a parthenolide-depleted the severity of experimentally induced polymorphous light
extract of feverfew, significantly reduced UV-induced ery- eruptions when applied 1 week prior to photoprovocation
thema in humans (64). with UVA in humans (73). However, since ferulic acid sig-
Additional studies are warranted to further clarify nificantly absorbs in the UVB/A-range, the photoprotection
whether the observed beneficial effects of those extracts or observed in those studies can likely not be attributed solely
their constituents cannot be at least partially attributed to their to its antioxidant properties (74). A significantly enhanced
potential sun-screening properties after topical application. antioxidative efficacy was also found for the combination of
α-tocopherol, ascorbic acid, and green tea polyphenols (75).
Thiol Antioxidants Studies revealed that the antioxidant synergism of this com-
Thiol antioxidants, such as GSH, N-acetylcysteine, lipoic acid, bination might be due to the regeneration of α-tocopherol by
and their derivatives are another important group of radical the green tea polyphenols, while the latter are regenerated
scavengers (1,2). Topical administration of GSH, GSH-ethyl by ascorbic acid.

Using an electron spin resonance spectroscopy–based wrinkles. Only agents which promote collagen, elastin, and
antioxidant assay (see next paragraph), a novel silicon-oil hyaluronic acid formation such as retinoic acid, human growth
based preparation comprising 15% L-ascorbic acid and 1% factors, and a few distinct peptides, have been shown to help
α-tocopherol combined with EGCG, the synthetic vitamin reverse the signs of skin aging.
E analogue dimethylmethoxy chromanol, and creatine was
shown to be significantly more effective in neutralizing free
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Topical Retinol: An Efficacious Solution for

Improvement of Main Photodamage Signs
Christiane Bertin and Thierry Oddos

INTRODUCTION of the molecule to form retinal, which is in turn reduced to

Clinically, skin aging is associated with a variety of signs retinol and esterified into retinyl ester. Retinyl esters are trans-
such as wrinkles, uneven pigmentation, skin roughness, skin ported and stored in the liver, which is the main storage site in
color and laxity. These clinical features are consecutive to the the body. The retinol is then delivered in the bloodstream asso-
structural and metabolic changes that occur during the pas- ciated with a specific transporter, the retinol-binding protein
sage of time (chronological aging). However, external factors (RBP). In normal conditions, most of the circulating retinol is
such as repeated skin exposure to solar ultraviolet (UV) radia- associated with RBP and the level of retinol in healthy volun-
tion can induce premature or photoaging of the skin. These teers fluctuates above 30 µg/mL (4).
changes are consecutive to a decrease in fibroblast number In the skin, at the keratinocyte level, retinol uptake is
as well as collagen synthesis and an increase in UV-induced mediated through the binding of RBP-retinol complex to spe-
collagen degradation by matrix metalloproteinases (MMPs). cific receptor(s), such as the 61 Kd RPE protein (retinal pigment
An accumulation of non-functional elastin is also observed in epithelium protein) and internalization (5,6,7).
the dermis, leading to a loss of skin elasticity and firmness. In In keratinocytes, retinol is stored in the form of retinyl
addition, a constant hallmark of skin aging and photoaging is esters (i.e., mainly palmitate, oleate, and acetate esters) (8,9)
epidermal thinning, which is triggered by a decrease in kera- primarily through the action of lecithin-retinolacyl transferase
tinocyte turnover rate (1). Photoaging is also associated with (10). It is then oxidized into retinal and retinoic acid by dif-
a dysregulation in melanin synthesis and distribution and a ferent alcohol dehydrogenases (11). Two other proteins respon-
general increase in the inflammatory status of the skin leading sible for retinol transportation play an important role in retinol
to the appearance of brown spots, a global increase in skin red- metabolism. The first is the cellular retinol-binding protein
ness, and telangectasia. (CRBP), which shows a high affinity for all-trans retinol. The
Retinoids have shown beneficial effects in reducing skin second is the cellular retinoic acid binding proteins (CRABP
photoaging. For instance, it has been demonstrated that all- I and CRABP II) that bind specifically retinoic acid. In skin,
trans retinoic acid (ATRA) improves skin photoaging signs CRABPII is the predominant retinoic acid binding protein and
(2). This clinical efficacy was mainly attributed to the effect of its expression is enhanced by keratinocyte differentiation and
ATRA on collagen metabolism by stimulation of the collagen treatment with retinoids. The role of these proteins is not com-
synthesis that ultimately accumulates in the upper part of the pletely clarified. CRBP could play a role in presenting the reti-
papillary dermis. Moreover ATRA downregulates UV-induced nol in the adequate conformation to the dehydrogenase. They
MMP1 and MMP9 expression (3), thereby replenishing collagen are also believed to control the level of free retinol and free
levels. Although ATRA is recognized as an effective therapy for retinoic acid and the translocation of these two molecules in
the treatment of photoaged skin through its regulatory effect on specific cellular compartments such as translocation of retinoic
collagen metabolism, it has been suggested that retinol (ROL), acid in the nucleus for CRABPII (12).
known as vitamin A, may also alleviate some major signs of
photoaging with a better irritation profile. Biological Effect on Skin Including
Retinoids are all derived from the naturally occurring on Different Cell Types
all-trans retinol. This molecule is composed of 15 carbons. Retinol plays a major role in the skin, as it is essential for regu-
Three parts of the molecule can be distinguished: a cyclic lating keratinocyte differentiation as suggested by the early
group, a polyene group, and a polar group (primary alcohol observation of abnormal keratinization in vitamin A–deficient
for retinol –CH2OH, and aldehyde –CHO for retinaldehyde rats (13) and humans (14). Topically, retinol stimulates the
and a carboxylic function –COOH for retinoic acid, the two proliferation of the basal keratinocytes through the release
latter compounds being obtained from the previous by oxida- of heparin-binding–epidermal growth factor (HB-EGF) from
tion [Figure 10.1]). Natural isomers carrying specific biological suprabasal keratinocytes (15,16). The influence of retinol on
activities have also been described as 11-cis retinaldehyde and keratinocyte differentiation is shown by the increase of some
9-cis retinoic acid. differentiation markers such as keratins (keratin 4, keratin
10, keratin 13, and cytokeratin 19) while others like fillagrine,
Metabolism of Retinol transglutaminase, or loricrine are decreased.
Diet is the normal source of retinol in the body. It origi- The overall effect on the epidermis is a stimulation of the
nates from carotenoids in plants and of the long-chain fatty cell proliferation in basal and suprabasal layers and an inhibi-
acid esters of retinol (as retinyl palmitate) in animal tissue. tion of the terminal differentiation of the keratinocytes. Taken
Carotenoids are split, in the intestine epithelium, in the middle together, this leads to a general thickening of the epidermis (8).

Although only slight clinical differences were observed, histol-

ogy showed a thickening of the previously atrophic epidermis,
decrease of keratinocyte dysplasia and atypia, more even dis-
persion of melanin, and formation of new dermal collagen and
blood vessels.
Since then, several clinical studies have been performed
(a) Retinol
to assess the activity of tretinoin on humans, especially in
the treatment of photoaging signs. Weiss and Voorhees in
H 1988 performed the first double-blind randomized placebo-
controlled study by applying 0.1% tretinoin on the forearm
and face of 30 patients for 16 weeks. They demonstrated a sig-
nificant improvement of photoaging in the tretinoin-treated
areas. The greatest improvement was in the reduction of fine
wrinkles, with a statistically significant difference occurring
(b) Retinaldehyde 8 weeks after beginning the study. Other features such as
coarse wrinkles, roughness, and sallowness also showed an
improvement, but to a lesser degree. Lever, in 1990, using
a preparation containing 0.05% tretinoin during 12 weeks,
O observed an improvement in clinical grading of photoaging
(fine lines and wrinkles). Grove again in 1991, assessing dif-
ferent concentrations of tretinoin (from 0.05 to 0.001%) over
(c) Retinoic acid 24 weeks, demonstrated a better efficacy of the dose activ-
ity with the highest concentration (0.05%) of the ingredient
Figure 10.1 Structure of most common retinoids (all trans on the Rz parameter (local mean amplitude) on skin replicas
conformation). (20). Olsen in 1992 on 296 subjects, with the same ranking
of concentrations as Grove, over a 24-week period showed
a decrease of skin roughness, hyperpigmentation, and fine
On dermal fibroblasts, retinol showed a potent stimu- wrinkling (21).
lation of the growth of fibroblasts from aged donors and a Unfortunately, tretinoin tends to be irritating, with many
stimulation of the synthesis of procollagen I and procollagen III patients experiencing redness, flaking, and increased skin sen-
(17). A suggested mechanism for the improvement of collagen sitivity. Some patients also reported increased sun sensitivity
synthesis by retinol involves the negative regulator of collagen during tretinoin therapy (22).
homeostasis, the cysteine-rich protein 61 (CCN1). Topical treat- Retinol, an alternative to tretinoin, has fewer side effects
ment with retinol for 7 days significantly reduced CCN1 mRNA and is a better tolerated additive in cosmetic preparations for
and protein expression in both chronologically and photoaged the improvement of photodamage signs. In 1995, Kang et al.
human skin in vivo (18). It has been shown that retinol could demonstrated that the application of retinol up to 1.6%, on
act also on elastin synthesis and elastin fiber formation (19). human skin under an occlusive patch, was unable to induce
Indeed, retinol treatment on dermal fibroblasts cultures as significant erythemal reaction versus vehicle (8). It also evokes
well as topical treatment on human skin explants resulted in the same physiological response as the 0.025% erythemato-
tropoelastin and fibrillin-1 gene expression and elastin fiber genic concentration of retinoic acid as demonstrated by epi-
formation as measured by qPCR and immunohistochemistry. dermal thickening and the enhancement of CRAPBII mRNA
Retinol represses the overexpression of UV-induced and protein expression. This retinoid activity of retinol was
MMP1 and MMP9, two enzymes involved in the degrada- obtained without a detectable increase of retinoic acid in the
tion of collagen molecules. The underlying mechanism is the epidermis. Duell et al. (23) showed that topical retinol pene-
repression by activated retinoic receptor of the of AP1 tran- trated the epidermis more and produced less irritation than
scriptional activity that drives the expression of MMP1, MMP3, the acid form. Finally, Goffin in 1997 tested 0.075% of retinol for
and MMP9. Skin exposure to UV light leads to the stimulation 8 weeks with corneosurfametry and demonstrated its better
of the MAP kinases such as ERK, JNK, or P38 and then to the tolerance versus tretinoin (24).
overexpression of the Jun protein which controls the level of
AP1 in the cell. Thus retinol is able to decrease the UV-induced Clinical Efficacy of Retinol
overexpression of MMPs and increase collagen synthesis, two Although retinol was demonstrated to induce in human
mechanisms that could explain in part its potential to protect skin a physiological response typical of retinoids, only a
the skin against photodamage (3). few clinical studies were published demonstrating its effi-
cacy in improving the signs of skin aging. In 2007, Kafi et al.
reported a clinical study to demonstrate the beneficial effect
CLINICAL ASPECTS of retinol on the skin signs associated with chronological
Retinoids have been widely studied during the past 20 years aging (25). This randomized study versus vehicle was per-
for their activity on human skin. In 1969, Kligman proved the formed on 23 elderly subjects over 80 years old (average 87
therapeutic efficacy of topical tretinoin (all-trans retinoic acid) years old). Retinol 0.4% and vehicle were topically applied
in acne vulgaris and later in 1986 pioneered the use of reti- on the upper inner arm for 6 months. The results showed a
noids in cosmetic dermatology by demonstrating its effects on significant improvement in fine wrinkles versus vehicle after
photoaged skin. He conducted an open study on eight elderly 2 months of application. This improvement increased with
patients receiving 0.05% tretinoin cream on the face for 6 to the continued application of retinol and was associated with
12 months, while six other patients received the vehicle alone. a significant better tactile roughness and overall severity

in chronologically aged skin. Skin biopsies demonstrated weeks vs. baseline) and then it appeared to reach a plateau.
a thickening of the epidermis and a significant increase Most importantly, the change in the fluorescence intensity
in GAG expression and procollagen I accumulation in the from baseline was significantly higher for the active-treated
dermis compared to the vehicle-treated arm. In this study, group than for the placebo-treated group at the 36-week
although rated as mild, adverse reactions were reported by time point (26). These anti-aging effects of retinol were con-
most subjects including erythema, peeling, pruritus, dry- firmed in two other shorter term double-blind randomized
ness, and burning or stinging, with the withdrawal of three studies performed with moisturizing product containing
subjects following severe enough symptoms (25). 0.1% retinol. In the first study the retinol- containing prod-
We explored the action of retinol at a lower concentra- uct was topically applied on 40 subjects (40–60 years old)
tion (0.1%) on a photoexposed area in order to investigate the once a day for 8 weeks. The effect of the product was com-
anti-aging effect of retinol at more tolerated doses on the pho- pared to the effect of its vehicle on 41 subjects. The results
toaging signs. In a first blind randomized vehicle-controlled presented in Table 10.1 showed that the application of the
study, 48 volunteers (41–60 years old) topically applied to the retinol 0.1% significantly improved the facial features of
face either the retinol 0.1% containing product or the vehicle, photodamaged areas as early as 4 weeks of use. Results
every day for 56 weeks. The clinical evaluation, performed from the second study have been published. In this 8-week,
by an expert grader after 12, 24, and 56 weeks of application, split-face, double-blind randomized clinical study, a 0.1%
showed that wrinkles under the eyes, fine lines at the crow’s retinol- containing moisturizer was tested (36 subjects) ver-
feet area, and skin tone evenness significantly improved sus placebo (28 subjects) in women with moderate facial
versus both baseline and vehicle. The improvement of the photodamage. Clinical evaluation of photodamage signs by
fine lines in the crow’s feet area was also demonstrated with a dermatologist demonstrated significant improvement ver-
digital imaging and with surface profilometry. A progres- sus placebo of fine lines, wrinkles, elasticity, firmness, and
sive improvement was seen between baseline and after 24 overall photodamage after 4 weeks. Thus, using a low level
and 36 weeks of treatment (Figure 10.2). The improvement of retinol (e.g. 0.1%) could still deliver a significant and rapid
in fine lines was also documented by surface profilometry, anti-aging efficacy while maintaining a good tolerance pro-
and a progressive disappearance of the fine lines in the file, as only a few subjects experienced a slight adverse skin
crow’s feet area was observed. Moreover, we demonstrated reaction without any withdrawal with the retinol product. In
that retinol stimulates epidermal cell proliferation along the two studies, the number of adverse reactions observed
with the treatment. The epidermal cell turnover rate was with the retinol product was not significantly higher than
evaluated using in vivo fluorescence spectroscopy by mea- with the vehicle. These clinical results were later confirmed
suring the fluorescence maximum attributed to tryptophan by two other clinical studies performed on middle-aged
moieties. The placebo-treated group did not show any sig- Japanese females (27). Both studies included 57 volunteers
nificant change from baseline for any time point. In contrast, and were performed with 0.075% and 0.04% retinol creams
the intensity of tryptophan fluorescence increased for the during 26 and 13 weeks, respectively. Both trials revealed
actively treated group evident from the first time point (12 significant improvement, especially on fine wrinkles, with

Figure 10.2 Example of improvement of crow’s feet area by retinol 0.1% product treatment.

Table 10.1 Clinical Assessment of Retinol Anti-Aging Efficacy

Vehicle Retinol 0.1%
Parameter 4 weeks 8 weeks 4 weeks 8 weeks
Under-eye wrinkles 0% 2.2% 4.3%* † 6.5%*†
Crow’s feet fine lines 8.1%* 10.8%* 29.7%*† 35.1%*†
Crow’s feet wrinkles 0% 0% 2.8%* 8.3%*†
Forehead wrinkles 0% 0% 6.4%*† 6.4%*†
Skin radiance 16.7%* 20.8%* 34.8%*† 43.5%*†
Cheek wrinkles 0% 0% 7.3%*† 9.8%*†
Overall photodamage 3.8%* 3.8%* 17.3%*† 19.2%*†

Note: Values represent the percentage of improvement versus baseline.

*Statistical significance versus baseline (p < 0.05).
†Statistical significance versus vehicle (p < 0.05).

minimal irritation. These results are very promising as they 5. Båvik CO, Peterson A, Eriksson U. Retinol-binding protein
demonstrate that retinol could be efficient at lower concen- mediates uptake of retinol to cultured human keratinocytes.
tration, thus decreasing irritation risks. Exp Cell Res 1995; 216:358–62.
Recently, we also demonstrated that prolonged use of 6. Hinterhuber G, Cauza K, Brugger K, Dingelmaier-Hovorka et
al. RPE65 of retinal pigment epithelium, a putative receptor
topical retinol (0.1%) significantly improved clinical signs of
molecule for plasma retinol-binding protein, is expressed in
photoaging on two 52-week, double-blind vehicle-controlled human keratinocytes. J Invest Dermatol 2004; 122:406–13.
studies (28). In the main study, 62 volunteers applied either 7. Huang J, Vieira A. Evidence for a specific cell membrane reti-
a stabilized retinol formulation or its vehicle to the full face, nol-binding protein transport mechanism in a human kerati-
while the second study evaluated histological/histochemical nocyte line. Int J Mol Med 2006; 17:627–31.
markers in 12 subjects after 52 weeks of either retinol or vehicle 8. Kang S, Duell EA, Fisher GJ, et al. Application of retinol to
use on contralateral dorsal forearms. The retinol group showed human skin in vivo induces epidermal hyperplasia and cellu-
significant photodamage improvement over vehicle at all time- lar retinoid binding proteins characteristic of retinoic acid but
points during the study. After 52 weeks, retinol had improved without measurable retinoic acid levels or irritation. J Invest
crow’s feet fine lines by 44% and mottled pigmentation by 84%, Dermatol 1995; 105:549–56.
9. Antille C, Tran C, Sorg O, Saurat JH. Penetration and metabo-
with over 50% of subjects showing +2 grades of improvement
lism of topical retinoids in ex vivo organ-cultured full-thickness
in many parameters. Additionally, at week 52, histochemical human skin explants. Skin Pharmacol Physiol 2004; 17:124–8.
data confirmed the clinical results, showing increased expres- 10. Törmä H, Vahlquist A. Vitamin A esterification in human
sion of type I procollagen, hyaluronan, and Ki67 expression (a epidermis: A relation to keratinocyte differentiation. J Invest
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CONCLUSION and Molecular Biology of Carbonyl Metabolism. New York: Kluwer
Skin aging is a complex process involving numerous differ- Academics/Plenum 1999; pp. 265–74.
ent mechanisms such as loss of cell proliferation, decreased 12. Roos TC, Jugert FK, Merk HF, Bickers DR. Retinoid metabolism
in the skin. Pharmacologica Rev 1998; 50:315–33.
potential of extracellular matrix synthesis, and overex-
13. Wolbach SB, Howe PR. Tissue changes following deprivation of
pression of matrix-degrading proteinases. Retinoids are
fat soluble A vitamin. J Exp Med 1925; 42:753–77.
naturally present in the skin and regulate epidermal cell 14. Frazier CN and CK Hu. Cutaneous lesion associated with
differentiation, activate collagen synthesis, and attenuate Vitamin A deficiency in man. Arch Intern Med 1931; 48:507–14.
the UV-induced overexpression of MMPs. The potential of 15. Chapellier B, Mark M, Messaddeq N, et al. Physiological and
regulating several pathways involved in skin chronologi- retinoid-induced proliferations of epidermis basal keratino-
cal and photoaging makes them good candidates as active cytes are differently controlled. EMBO J 2002; 21:3402–13.
ingredients to alleviate facial aging signs. Although retinoic 16. Rittié L, Varani J, Kang S, et al. Retinoid-induced epidermal
acid has been the first retinoid to demonstrate an efficacious hyperplasia is mediated by epidermal growth factor receptor
wrinkle improvement, the severe side effects induced by its activation via specific induction of its ligands heparin-binding
EGF and amphiregulin in human skin in vivo. J Invest Dermatol
topical application warrant the identification of other reti-
2006; 126:732–9.
noids for this benefit. Therefore retinol was identified as a
17. Varani J, Warner RL, Gharaee-Kermani M, et al. Vitamin A
good alternative to retinoic acid, as its topical application antagonizes decreased cell growth and elevated collagen-
induces a low level of skin adverse reaction while delivering degrading matrix metalloproteinases and stimulates collagen
physiological skin responses similar to those induced by ret- accumulation in naturally aged human skin. J Invest Dermatol
inoic acid. Moreover, the retinoic acid level in the epidermis 2000; 114:480–6.
observed after topical application of retinol is extremely 18. Quan T, Qin Z, Shao Y, et al. Retinoids suppress cysteine-rich
low  or undetectable and thus provides a substantial mar- protein 61 (CCN1), a negative regulator of collagen homeostasis,
gin of safety with respect to possible systemic effect. Results in skin equivalent cultures and aged human skin in vivo. Exp
presented here focus preferentially on the clinical efficacy of Dermatol 2011; 20:572–6.
19. Rossetti D, Kielmanowicz MG, Vigodman S, et al. A novel anti-
well-tolerated doses of retinol. Indeed, in two double-blind
ageing mechanism for retinol: Induction of dermal elastin syn-
randomized placebo-controlled studies, retinol at 0.1% dem-
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features such as fines lines and wrinkles within 1 month’s photodamaged skin after therapy with tretinoin emollient
time. cream. J Am Acad Dermatol 1991; 25:231–7.
21. Olsen EA, Katz HI, Levine N, et al. Tretinoin emollient cream: A
new therapy for photodamaged skin. J Am Acad Dermatol 1992;
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Applications of Non-Denatured Soy in Skin Care

Jue-Chen Liu, Jeff Wu, and Miri Seiberg

INTRODUCTION as meat and dairy substitutes in various items. Today, most

The soybean plant belongs to the pea family, Leguminosae, people are aware of the use of soy proteins in baby formula,
subfamily Papilionoidezae, and the genus Glycine. The culti- weight-loss drinks, sport drinks, and as a low-fat substitute
vated form is named Glycine max (L.) Merr. It is one of the old- for hamburger. Soybeans are also valued for their medicinal
est annual crops of the Far East. Dietary soy has been known qualities. Epidemiological data indicates that people from
for centuries to provide nutritional, medicinal, and health ben- Asian cultures have lower rates of certain cancers, including
efits. Because of the nutritional value of soybeans, there is an cancers of the breast, prostate, and colon, and lower rates of
inherited value for skin care applications. In fact, the use of soy cardiovascular disease, osteoporosis, and postmenopausal
to treat skin conditions has a long history. It was first reported symptoms (10–16). In the past decade, studies on soy’s health
in Traditional Chinese Medicine Encyclopedia (1) that soy- benefits have dramatically increased in these areas, and more
beans provided therapeutic efficacy against skin conditions systematic and controlled clinical studies have been presented
including hyperpigmented lesions and dry skin. Additionally, on its health benefits (17–18). In 1999, the U.S. Food and Drug
Chinese folklore has it that women workers in tofu industries Administration (FDA) approved a healthy claim for soy pro-
have the most beautiful skin, i.e., fair, smooth and porcelain- tein that associated with reduced risk of coronary heart dis-
like fine skin. For a very long time, soy or components of soy ease (19). This ruling is based on the FDA’s decision that foods
for topical applications have been limited to functional appli- containing soy protein included in a diet low in saturated fat
cations such as emollient and skin conditioning agents. The and cholesterol may reduce the risk of coronary heart disease
scientific fundamentals and clinical efficacy of soy for skin ben- by lowering blood cholesterol levels.
efits have not been proven until recent years. It was reported
that soy isoflavones possess phytoestrogenic properties which
may play a role in the skin of menopausal women (2). Specific
protein components in soybeans were identified for inhibition
For many years, due to the lack of basic research in under-
of melanosome transfer resulting in pigment reduction (3–5).
standing the fundamental mechanisms of soy on skin, soy’s
Other soybean components such as lipids, oligosaccharides,
nutritional components were used mainly as functional ingre-
and saponins can also provide skin benefits. Such new knowl-
dients in the cosmetic industry (20) and mostly have remained
edge and findings have been implemented into dermatological
so until today. The most frequently used soy components
and cosmeceutical products for broader skin applications. This
include soybean oil, soy protein isolates, and soy lecithin
chapter reviews the components of soy and their clinical uses
as shown in Table 11.1. Soybean oil is one of the valuable
in improving skin conditions.
materials used to adjust the rheology of cosmetic emulsions.
Examples of soy oil as an emollient and moisturizer include
SOY IN HUMAN HEALTH baby oil and moisturizing lotion. Soy protein isolate/hydro-
Soybeans originate from China. In 2853 BC, Emperor Sheng- lysate has been formulated as an emulsifier and foaming
Nung of China named five sacred plants—soybeans, rice, enhancer into a wide variety of products, including laundry
wheat, barley, and millet. Soybean plants were then domesti- detergent, bath products, shampoos, and skin cleansers (21).
cated and cultivated into a food crop for everyday diet (6–7). The lecithin fraction of crude soybean oil has found applica-
The soybean has blossomed from legendary Chinese origins to tion as an emulsifying, surface active agent, and a stabilizing
the “miracle crop” vastly produced on modern-day American agent in industrial products such as paint, ink, soap, and cos-
farms since its first introduction into the United States in the metics (22). Soy sterols are employed as skin conditioners for
early 1800s (7). lotion, creams, and cleansers (23).
From about the first century AC to the Age of Discovery In the early 1990s, soy extracts or soy germ extracts
(15th to 16th century), soybeans were introduced into several that claimed to provide specific biological activities started
countries such as Japan, Indonesia, the Philippines, Vietnam, to appear in skin care products. These soy extracts, although
Thailand, Malaysia, Burma, Nepal, and India. The first soy- defined vaguely as soy (soja) concentrates or soy proteins,
beans arrived in America in the early 1800s and since then were actually produced using different chemical extraction
have played an increasingly important role for human nutri- conditions and could result in dissimilar compositions and
tion and health. skin effects. Such soy extracts often had too small a fraction
Soybeans with their valuable proteins and oil are an of the original soy components to provide the desired biologi-
important source of nutrition. The soybean is rich in miner- cal activities to keep the bioactivities for skin efficacy, or were
als including calcium, iron, and potassium, amino acids, and processed excessively under harsh extraction conditions such
vitamins. It is also a good fiber source (8–9). They can be used as extreme pH, strong solvents, and elevated temperatures.

Table 11.1 Traditional Use of Soy in Skin and Hair Care

Soy components Functionality Applications Reference
Soybean oil Skin conditioner/ emollient Baby oil, (22)
Soy protein Isolate/hydrolyzate Emulsifier Bath products, cleansers, (21)
Soy lecithin Surface active, emulsifier, Liposome (60)
/phospholipids stabilizing agent

Soy sterol Skin conditioner Lotion /cream/cleanser (20)

Few clinical reports or publications were available for cosmetic deposition. This effect is possible only when keratinocyte-
products spiked with such soy extracts. The use of these soy melanocyte contact is established (see Figure 11.1, top panel).
extracts has been limited to product marketing claims. Melanocytes alone do not respond to PAR-2 modulating agents
with pigmentary changes (3). When serine protease inhibitors
interfere with PAR-2 activation, melanosome transfer from the
SCIENTIFIC ADVANCES IN THE USE melanocytes to the keratinocytes is inhibited. The reduced
OF SOYBEANS FOR SKIN CARE melanosome ingestion by keratinocytes results in depigmenta-
Soy Proteins, Non-Denatured Soybean tion both in vitro and in vivo (3,38). This depigmenting effect
Extracts, and Depigmentation caused by serine protease inhibitors is reversible in vivo (3,38),
There is an increasing global desire for skin care products to therefore excluding the possibility of melanocyte death follow-
induce fair skin and even skin tone. In addition, hyperpig- ing such treatments.
mented spots including mottled hyperpigmentation, solar In soy foods, STI and BBI are inactivated and denatured
lentigines, and melasma are key concerns for the aging popu- by heat and pressure or by fermentation to prevent the block-
lation and pregnant women. The most frequently used topical ing of trypsin activity in the digestive system for human,
treatment agents for depigmentation are tyrosinase inhibitors, which would cause gastrointestinal adverse effects.
hydroxyacids, retinoids, hydroquinones, anti-inflammatory It was found that non-denatured soybean extracts, non-
agents, and their combinations. Treatment with these agents is denatured soy, and the isolated soy- derived proteins STI and
not always satisfactory and may result in irritation, sun sensi- BBI inhibit PAR-2 activation, resulting in skin depigmenta-
tivity, or toxicity (24). In its proposal published in the U.S. gov- tion (5). As shown in Figure 11.1, keratinocyte-melanocyte
ernment’s Federal Register, U.S. FDA notes: “The actual risk to co-cultures treated with the soy protein STI have reduced
humans from use of hydroquinone has yet to be fully deter- tyrosinase activity, documented by DOPA staining (upper
mined…. We’re acting for safety reasons. There is a potential panel). Darkly-pigmented human skin grafted on immuno-
for hydroquinone to be a carcinogen in humans.” In December deficient mice and treated with STI showed reduced pigment
2009, U.S. FDA nominated hydroquinone for further study by deposition, as demonstrated by Fontana-Mason (F&M) stain-
the National Toxicology Program (NTP). FDA believed that the ing of histological sections after five weeks of topical treatment
NTP studies are necessary before it can determine whether (middle panel). F&M staining identifies silver nitrate–reducing
hydroquinone is generally recognized as safe (GRAS) when molecules. In skin, this nonspecific stain identifies primarily
used in over-the-counter (OTC) skin bleaching drug products. melanin. Dark-skinned swine treated with the non-denatured
Therefore, safer and more effective depigmenting agents are soy extract displayed visible skin lightening following 8 weeks
needed to address consumer needs for fair skin, even tone, of treatment (lower panel). Therefore, agents such as STI, BBI,
and spot reduction. Moreover, a move toward “green” thera- or non-denatured soybean extracts may serve as alternative,
pies has led to a demand for natural, safe, and efficacious skin- natural treatments for skin lightening or hyperpigmentation.
lightening agents.
Soybeans contain two proteins, which are reported to
inhibit the protease-activated receptor-2 (PAR-2) pathway, Non-Denatured Soybean Extracts
leading to reduced melanosome transfer from melanocyte to and Delayed Hair Growth
keratinocyte and resulting in pigmentation reduction. These Mammalian hair provides environmental protection, but this
two proteins are serine proteinase inhibitors: the Kunitz- function has been lost in humans, who keep or remove their
type  trypsin inhibitor (soybean trypsin inhibitor [STI]), and hair at different body locations for social and cosmetic pur-
the Bowman-Birk protease inhibitor (BBI) (25–26). STI has a poses. Many procedures are used to remove unwanted hair,
molecular weight of 20 k daltons (k-Da), consists of 181 amino ranging from simple and inexpensive treatments like shaving
acid residues with two disulfide bridges, and is roughly spher- to costly and time-consuming methods such as electrolysis and
ically shaped (27). BBI is an 8 k-Da protein that inhibits the pro- laser therapies. Hair removal methods differ in the duration of
teases trypsin and chymotrypsin at separate reactive sites (28). the effect, pain and discomfort levels, and possible undesired
PAR-2 is a G-protein-coupled receptor activated by a serine side effects (39).
protease cleavage (29–31). Trypsin, mast cell tryptase, and kal- The hair follicle undergoes cycles of active growth (ana-
likrein are the only known natural serine proteases that acti- gen), regression (catagen) and rest (telogen) (40). While the
vate PAR-2 (32–34). PAR-2 is expressed in keratinocytes (35–36), morphological changes throughout the hair cycle are well doc-
but not in melanocytes (3) and is involved in the regulation of umented (41), the regulation of the different phases of this cycle
pigmentation (3,37–38). It was reported that the inhibition of is not completely understood. It was recently demonstrated
PAR-2 activation by serine protease inhibitors reduces pigment that non-denatured soybean extracts and the soybean-derived

(a) (b)

(c) (d)


Figure 11.1 The inhibition of PAR-2 activation by serine protease inhibitors results in reduced pigment deposition. Keratinocyte-
melanocyte co-cultures treated with the soy protein STI have reduced tyrosinase activity, documented by DOPA staining (Top panels
(a) Control vs. (b) Treated). Hyperpigmented human skin grafted on immunodeficient mice and treated with STI showed reduced pigment
deposition, demonstrated by F&M staining of histological sections after 5 weeks of treatment ([c] control vs. [d] treated). Dark-skinned
swine treated with a non-denatured soy extract demonstrates visible skin lightening following 8 weeks of treatment (e).

proteins STI and BBI reduce not only hair pigmentation but skin produces higher amounts of prostaglandin E2 (PGE2, the
also the rate of hair growth and the final dimensions of the product of the COX-2 pathway) relative to young skin (52). In
hair shaft (5). Following non-denatured soy treatment, the animal models, oral and topical COX-2 inhibitors have chemo-
onset of anagen was delayed, the duration of the hair cycle was preventive activity against UV-induced skin cancer (reviewed
reduced, and the resulting hair shafts were shorter, thinner, in 50, 51). COX-2 expression is reduced in mouse skins upon the
and softer. As shown in Figure 11.2, mouse hair treated with topical applications of soy isoflavones (53).
non-denatured soy showed reduction in the length of the hair Non-denatured soybean extracts inhibit the COX-2 path-
shafts (upper panel). Hair follicle development (anagen) was way (54). Cultured keratinocytes or epidermal equivalents
delayed as seen following 7 days of treatment (middle panel). were pretreated with non-denatured soybean extracts, washed,
While the untreated follicles were completely developed, at and then UV-exposed. As shown in Figure 11.4, UV irradia-
this time point the soy-treated follicles were smaller and con- tion induced COX-2 expression and PGE2 secretion, which were
tained reduced levels of pigment. At day 15 of treatment, when reduced upon pretreatment with the soy extracts (54). When mice
all follicles had reached their final dimensions, the soy-treated were topically treated with non-denatured soybean extracts,
follicles were significantly smaller (lower panel). While the soy then washed and then UV-exposed, they had reduced levels of
isoflavones may play some role in the delayed hair growth by COX-2 expression in their skins as compared to mice exposed
the non-denatured soy lotion, the ability of the non-denatured to UVB without soy pre-treatment (54). These data confirm the
soy product to thin the hair and facilitate less-frequent hair COX-2 inhibitory activity of the non-denatured soybean extracts
removal seems to result primarily from the actions of two the in vivo and suggest their possible activity in delaying photoag-
soy-derived proteins STI and BBI (5). These results indicate ing symptoms and skin cancer processes in human skin.
that agents such as non-denatured soy extracts may serve as
an inexpensive, natural alternative treatment for undesired Non-Denatured Soybean Extracts Reduce Skin
hair growth. Cancer Progression in High-Risk Mice
Polyphenols, including green tea polyphenols, resveratrol, and
soy isoflavones like genistein, have a photoprotective effect on
Non-Denatured Soybean Extracts UV-induced skin inflammation, oxidative stress, and DNA dam-
and Dermal Matrix Enhancement age (55). The soy protein BBI is chemoprotective, and an organic
Skin elasticity and elastin fiber production are reduced with extract of soybeans enriched in BBI has been evaluated in clini-
aging (42–44) as elastin synthesis decreases and the secretion cal studies (26). Mice fed with a non-denatured soymilk pro-
of elastases increases. UV exposure induces extensive elastin tein supplement had a reduction in the numbers and volumes
synthesis and crosslinking (“solar elastosis”), which add to of experimentally-induced skin tumors (56), however this was
skin’s rigidity. Sunscreens, antioxidants, and retinoids are pro- accompanied with undesired gastrointestinal effects. Since non-
moted as “antiaging” agents, however they are not specific for denatured soybean extracts contain isoflavones and BBI, and
repairing or enhancing skin elasticity. Inhibiting UV-induced since they possess COX-2 inhibitory activity, the use of these
fibroblast-derived elastases protected the dermal elastic net- extracts as topical chemopreventive agents was evaluated.
work and reduced wrinkle formation in rodents (45–46). The Hairless mice exposed to chronic low UV levels become
non-denatured soybean extracts were found to have elastase- “high-risk mice.” They are tumor-free, but with a high risk of
inhibitory activities (47), to induce the synthesis of both col- developing skin tumors in the absence of additional UV expo-
lagen and elastin, and to promote the correct assembly of sure. High-risk mice were topically treated with non-denatured
newly-synthesized elastin fibers, suggesting both dermal pro- soymilk, heat-denatured soymilk, STI, or BBI, or with water or
tection and restoration of the dermal extracellular matrix. BSA as controls (57). Non-denatured soybean extracts, but not
Human skin, transplanted onto immunodeficient mice heat-denatured soybean extracts, reduced the incidence and
and topically treated with these extracts, showed an increase slowed the growth and progression of skin tumors (see Figure
in the expression of elastin, elastin-accessory proteins, and 11.5). STI and BBI had a similar but reduced effect, suggesting
collagen (47). Human skin explants treated ex-vivo with non- that other components of the non-denatured extract provide
denatured soybean extracts showed an increase in collagen, additional protection. The number of tumor-bearing mice,
elastin, and fibrilin-1 production by histological staining and tumors per mouse, and the volume of the visible part of the
immunohistochemistry (see Figure 11.3). These data suggest tumors were all markedly reduced upon the topical treatment
that non-denatured soybean extracts could induce collagen of non-denatured soybean extracts (57). Histopathological
and elastin synthesis in human skin and provide cosmetic examination documented numerous squamous cell carci-
antiaging effects. noma (SCC) lesions in the UV-exposed mice. The non-dena-
tured soybean extracts-treated skins, in contrast, had very
Non-Denatured Soybean Extracts May small lesions, with no dysplasia or carcinoma, which were not
Reduce the Risk of Non-Melanoma classified as SCC (Figure 11.5). These data might suggest that
Skin Cancer Development non-denatured soybean extracts could be topically applied to
Non-Denatured Soybean Extracts Inhibit the COX-2 Pathway humans to reduce or prevent UV-induced skin damage and to
Chronic inflammation is associated with numerous skin con- reduce the risk of skin tumor formation and progression.
ditions and diseases, ranging from skin aging to epithelial
skin tumors (reviewed in 48, 49). Cyclooxygenase-2 (COX-2) is NON-DENATURED SOY FOR COSMETIC
involved in UV-induced skin inflammation and carcinogen- DERMATOLOGY APPLICATIONS
esis (reviewed in 50,51), and in UV-induced edema, epidermal Non-Denatured Soy Composition
hyperplasia, and the generation of oxidative DNA damage. While the compositions of natural soybeans can inevitably
Repeated UV exposure results in chronic upregulation of be subject to variations, depending on the particular harvest,
COX-2 expression and chronic inflammation, which accelerate soil/region, and species variation, the basic chemical composi-
skin aging and increase skin cancer risk. Indeed, aged human tion is similar, as illustrated in Table 11.2 (58). Mature soybeans

(a) (b)

(c) (d)

(e) (f )

Figure 11.2 Images of hair growth patterns under different conditions. (a) Untreated control and (b) treated illustrate reduction in the
length of the mouse hair shafts treated with a non-denatured soy extract. (c) and (d) show delayed hair follicle development anagen) as
seen following 7 days of treatment. While the untreated follicles (c) were completely developed, at this time point the soy-treated follicles
(d) were smaller and contained reduced levels of pigment. At day 15 of treatment, when all follicles had reached their final dimensions,
the soy-treated follicles were significantly smaller (e and f).

(a) (b)

(c) (d)

(e) (f )

Figure 11.3 Non-denatured soybean extracts induce collagen and elastin synthesis. Human skin explants (obtained from surgical
procedures with informed consent and institutional board approval) were maintained in culture for 8 days, with or without topical daily
treatment, 5 days/week, using a base formulation with 2.5% non-denatured soybean extract. (a) Untreated control versus (b) treated
Herovici staining documents mature collagen fibers (magenta-red). Topical treatment with non-denatured soybean extracts induces
the production of collagen fibers. (c, d) Luna elastin staining documents mature elastin fibers (purple-brown). Topical treatment with
non-denatured soybean extracts enhanced the ewlastic fiber network. (e, f) Immunohistochemical staining documents tropoelastin (the
elastin fiber monomer, green) and fibrillin-1 (an elastin-accessory protein, red). Non-denatured soybean extracts induce tropoelastin and
fibrillin-1 synthesis, and their co-localization (yellow) documents the site of elastin fiber formation. ([a,b] Images courtesy of Dr. CB Lin
and D. Rossetti.)

PGE2 (pg/mL)



Control UVB Soy+UVB
(a) (b)

(c) (d)

Figure 11.4 Non-denatured soybean extracts inhibit the COX-2 pathway. Epidermal equivalents were topically treated for 24 hours with
2% non-denatured soybean extracts in PBS or with PBS alone, and then UVB-exposed (100 mJ/cm2) as in (ref. 54). Samples were col-
lected at 24 hours post UVB exposure for analysis. (a) Culture media were analyzed for PGE2 secretion. Non-denatured soybean extracts
inhibited the UV-induced secretion of the inflammatory mediator PGE2. (b–d) COX-2 staining of the treated equivalents: (b) PBS, (c) PBS
followed by UVB, (d) 2% non-denatured soybean extracts followed by UVB. Non-denatured soybean extracts led to reduced COX-2 pro-
tein levels after UV exposure.

are composed of up to 36% proteins, 30% carbohydrates, and for human skin care applications. However, these processes
20% lipids. The highly innovative non-denatured soy technol- frequently denature the active proteins in the soy, resulting in
ogy was created based on scientific findings. The non-dena- a compromised biological efficacy, e.g., a reduction in prote-
tured soy raw material was created from whole soybeans ase inhibitory activity. Furthermore, such processes also can
without using either chemicals or chemical solvents. The novel lead to instability of the soy product as well as to an undesir-
standardization of the manufacturing process results in a able odor and color generation. A novel manufacturing pro-
blend of balanced non-denatured components including active cess was developed for the non-denatured soy technology to
proteins, essential lipids, oligosaccharides, and other soy reduce the levels of microbial contamination in non-denatured
ingredients, which are in a similar proportion to that naturally soy and maintain its protease (trypsin) inhibitory activity and
present in the soybeans. The standardization process includes preserve stability. This proprietary gamma irradiation process
quality control such as measurements and lower limits of bio- with optimized intensity and duration enabled reducing bio-
chemical activities, of serine protease inhibitory activities. burden to attain safe levels while maintaining a stable non-
It is well known that due to its natural origin, high denatured composition with full protease inhibitory activity,
levels of microorganisms are commonly associated with soy- and with no color or odor changes.
beans and soy products. Consequently, decontamination pro- Table 11.3 illustrates the non-denatured soy components
cesses such as heat, organic/aqueous solvent extraction, and and their potential applications in skin care. Non-denatured
high shear purification are used to reduce microorganism con- soy contains both non-denatured small and large soy proteins.
centrations in soy products, to allow soy ingredients to be safe The small soy proteins include STI, BBI, and lunasin, with their

Control Largest soy-treated lesion

(a) (b)


Heated soymilk

Water control
Tumors per mouse

2 control


Average untreated tumor

0 6 9 12 15 18 21
(c) (d)

Figure 11.5 Non-denatured soybean extracts slow the progression of skin cancer in high-risk mice. SKH-1 mice were irradiated with
UVB (30 mJ/cm2) twice weekly for 20 weeks and then UVB irradiation was stopped (as in ref. 57). Three weeks later, the mice were topi-
cally treated once a day, 5 days/week, for 21 weeks, with non-denatured soymilk, heat-denatured soymilk, or water control. (a) H&E stain-
ing of visible skin lesions and visually unaffected skins (same magnification): Control skin (not exposed to UV and not treated). (b) Largest
visible lesion of a non-denatured soymilk-treated skins. (c) An average lesion in the high-risk, UV-exposed and untreated group shows a
well-differentiated SCC. Topical treatment with non-denatured soymilk significantly reduced the severity of the developing skin lesions.
(d) The number of visible lesions per mouse was determined every 3 weeks. Topical treatment with non-denatured soymilk, but not with
heat-denatured soymilk, led to a marked reduction in the number of tumors per mouse. (Images courtesy of C. Paine.)

Table 11.2 Soybean Composition Characteristics

Major components Green/Raw % wt/wt Mature seeds % wt/wt
Water 67 8
Proteins 12 36
Lipids 6 19
Carbohydrates 11 29
Fiber 4 8

Source: USDA National Nutrient Database for Standard Reference, 1999. Available at:

Table 11.3 Non-Denatured Soy Components with Potential Applications in Skin Care
Soy components Skin care applications Reference(s)
Proteins Small soy proteins (STI, BBI, Depigmenting and delay hair (3–5, 37–38, 63)
30%–50% Lunasin) growth
Large soy proteins (e.g. Skin softening and smoothing (60)
glycinin 360 k-Da,
β-conglycinin 180 k-Da)
Lipids Essential fatty acids (linoleic, Antioxidant protection, skin (60–62)
10%–30% linolenic, oleic acids) lightening, restore barrier
Lecithins/ phospholipids Skin moisture, cleansing (60)
Carbohydrates Di- and oligosaccharides and Skin hydration (60)
Minor components Soy flavones Weak antioxidant and anti- (64–67)
(e.g. Daidzein, genistein, inflammatory, inhibition of
daidzin, genistin tyrosine
Phytosterols Skin moisture, (68–69)
Vitamins (tocopherols) Antioxidant (60)
Minerals Deficient can cause untoward (71)
Saponins Cleansing (60, 72)
Others Phytic acid Depigmentating (60)

depigmenting activity retained and quantified (3–4). Unlike Non-denatured soy also contains minor components
trypsin inhibitors of soy protein nature, the non-protein tryp- like isoflavones, vitamins, and minerals, which could offer
sin inhibitors lack specificity and are very susceptible to cat- important skin care benefits. Isoflavones have been reported
ionic suppression (59). The large proteins of the non-denatured to provide a number of skin care benefits. They have weak
soy extract include hundreds of proteins with varying molecu- antioxidant and anti-inflammatory activities, which are
lar weight up to millions Daltons. The most abundant proteins important to combat oxidative stresses induced by the sun.
are glycinin and conglycinin, with molecular weight around A number of studies have indicated isoflavones can prevent
180 k-Da and 360 k-Da (60). Each protein group precipitates and treat sun-induced cancer (64–65). Isoflavones such as
at different pH values, which may affect topical formulation genistein and daidzein, normally classified as phytoestro-
gens, originate in plants, unlike estrogens, which are of ani-
preparations. Non-denatured soy contains essential fatty
mal origin. The isoflavone structures are nonsteroidal, while
acids, including linoleic acid and linolenic acids, which are
all estrogenic hormones are steroidal (66–67). Soybean oil can
essential to restore stratum corneum barrier functions and
contain as much as 0.37% of phytosterols, which are mostly
may potentially help acne resolution. These unsaturated fatty
in unsaponifiable forms (68). The most significant phytos-
acids may also provide antioxidant properties (61). In addition, terols found in oils and fats are campesterol, stigmasterol,
linoleic acid is known to enhance skin penetration of various beta-sitosterol, delta 5-avennasterol, and delta7-stigmasterol.
compounds (62). Crude soybean oil contains 1%–3% phospho- Phytosterols are reported to provide anti-inflammatory
lipids, which are the major components of cell membranes (63). effects via the arachidonic cascade. Clinical applications
Phospholipids can also form liposome, a vesicle delivery sys- can be found for antipruritic effects, reducing sun-induced
tem that could enhance skin penetration for both hydrophilic erythema, and diaper dermatitis (68–69). Such characteris-
and lipophilic large molecules such as proteins, and small tics combined with the conditioned use make them possible
such as alpha-hydroxy acids. Non-denatured soy contains alternatives to cortisone and corticosteroids, as well as sili-
about 30% carbohydrates, which can provide skin hydration cone replacement. Soybeans contain both water-soluble and
properties. oil-soluble vitamins. The water-soluble vitamins present in

soybeans mainly include thiamin, riboflavin, niacin, pan- formulas does not adversely affect human growth, develop-
tothenic acid, and folic acid. The oil-soluble vitamins found ment, or reproduction (78).
in soybeans are vitamin A and E. Soybean oil contains a The non-denatured soy products contain much lower
substantial amount of vitamin E, which provides effective concentrations of isoflavones, typically less than 0.01% soy
natural antioxidant and nutrition values (70). Soy contains isoflavones, which is negligible compared to the level of phy-
relatively high levels of desired metal ions such as iron, zinc, toestrogen intake by average consumers in most Western
manganese, magnesium, and calcium that are important in countries. By way of example, following European guidelines,
skin health. Metal ions play an important role in skin health. the estimated maximum exaggerated topical exposure from
Deficiency in certain metal ions in the skin will result in non-denatured soy products (0.96 mg/day) is considerably
unwanted effects such as dermatitis, discoloration of hair, lower than the measured dietary intake for mid-life Australian
and retardation of hair growth. Optimal metal ions such women (~ 17 mg/day) (79), which is, in turn, less than the
as manganese, magnesium, calcium, and zinc are needed exposure by infants fed soy-based formula milk (22–45 mg
to maintain skin health (71). Soy saponins (2,3-dihydro-2, /day) (80). Moreover, the potential systemic isoflavone expo-
5-dihydroxy-6-methyl-4H-pyran-one) contain carboxyl sure from the non-denatured soy products (assuming 60 kg
groups, which have surface activity for foaming and emul- body weight, 0.00208 mg/kg/day = 2.08 ng/g/day) is insignifi-
sifying power in cosmetic formulation (72), and are also cant when compared to typical systemic concentrations. For
reported to provide an active oxygen-scavenging effect in example, the endogenous systemic plasma baseline concentra-
the skin and maintain healthy epidermal conditions (73). tion for U.S. adult women is 7.94–34.54 ng/mL (81) and for UK
adult women consuming 20 g roasted soybean products, the
Safety of Non-Denatured Soy for systemic concentration is 540ng/mL genistein (82).
Human Topical Applications Broadly speaking, however, a rank order may be deter-
It is known that some naturally derived ingredients can elicit mined of daily isoflavone intake: soy formula fed infant
irritant or allergic responses. Soy has been used for centuries (approximately 40 mg/day), average Japanese consumer
and is well known to be safe for human use. A systematical (approximately 25–100 mg/day), vegetarian consumer (approx-
safety evaluation has been completed to determine the clini- imately 3 mg/day), average consumer (approximately 1 mg
cal topical profiles of the non-denatured soy raw material and /day) (109). There is no data documenting that soy extract or
the non-denatured soy-containing skin care formulations (74). soy oil has estrogenic effects when applied to the skin as it
Clinical cumulative irritation evaluations utilized two dif- might when taken orally at high doses (83).
ferent methodologies that consisted of patching the subject A new longer-term (2 years), multicenter, randomized,
with the formulation over a 2–3 week time period. Method A double-blind, placebo-controlled clinical trial assessed the
was conducted to assess irritancy, in which a minimum of 25 effects of daily high level of supplementation with 80 or 120 mg
subjects was patched continuously for a total of six applica- aglycone equivalent soy hypocotyl isoflavones plus calcium
tions over a 2-week period. Method B was conducted to assess and vitamin D on the health of 403 postmenopausal women
comparative irritation potential of the formulations, in which (84). At baseline and after 1 and 2 y, clinical blood chemistry
approximately 200 subjects were patched intermittently with values were measured and a well-woman examination was
the formulation three times a week for a total of nine appli- conducted, which included a mammogram and a Papanicolaou
cations over a 3-week period. All test sites were graded three test. A cohort also underwent transvaginal ultrasound mea-
times a week after patch removal. Using a scale of potential surements to assess endometrial thickness and fibroids. The
total irritation score of 660, both non-denatured soy and all baseline characteristics of the groups were similar. After 2  y
skin care formulations with soy resulted in negligible cumula- of daily isoflavone exposure, all clinical chemistry values
tive irritation score <10. Comparative cumulative irritation of remained within the normal range. Isoflavone supplementa-
non-denatured soy and vitamin formulas also demonstrated tion did not affect blood lymphocyte or serum free thyrox-
negligible irritation. Non-denatured soy formulations were ine concentrations. No significant differences in endometrial
clinically evaluated with the repeated insult patch test (RIPT). thickness or fibroids were observed between the groups. It
These studies demonstrated that specific topical formula- concluded that daily supplementation for 2 y with 80–120 mg
tions containing the non-denatured soy alone, soy extract, soy hypocotyl isoflavones has minimal risk in healthy meno-
or soy extract in combination with vitamins A and C did not pausal women.
induce contact dermal sensitization. Photosensitivity was also A placebo controlled in-vitro dermal absorption study
assessed by photoallergy and phototoxicity tests, which did using human cadaver skin found that the potential systemic
not find any photosensitivity when non-denatured soy was soy isoflavones absorption from topical applications of non-
applied. Recently, soy isoflavones have drawn attention for denatured soy products is below the detection limits of the
their potential phytoestrogenic effects. Soy isoflavones differ current state-of-the-art analytical instrumentation and neg-
significantly in terms of their molecular structure from estro- ligible compared to any endogenous levels of isoflavones
gens, such as estradiol, and they are not metabolized to these resulting from dietary soy consumptions or supplement con-
estrogens. Experimental data suggest that isoflavones exhibit sumptions of infant soy milk. The potential local epidermal
an estrogenic-like potency of between 1000 and 100,000 times and dermal accumulations for soy isoflavones are estimated
lower than estradiol, depending on the nature of the assay to be at least 2500 times lower than the no-observed adverse
used. (75–77). Largely as a result of research in in-vitro or in effect level (NOAEL) limit. In summary, soy isoflavones are
animal models, concerns have been voiced regarding isofla- naturally occurring in non-denatured soy at trace levels, and
vone use in soy products and soy infant formulas in relation their potential systemic absorption is negligible and presents
to nutritional adequacy, sexual development, neurobehav- no harmful risks to human health.
ioral development, immune function, and thyroid disease. Council of Europe guidance on the use of plants in
Available evidence from adult human and infant populations cosmetics specifically endorses the use of soy extracts. With
indicates that dietary isoflavone consumption in soy infant particular reference to phytoestrogens, guidance from 1994

provides that “the use of a natural ingredient in cosmetic prod- Collagen Production
ucts is recommended only if the finished products are free of Preclinical studies of a non-denatured soy composition were
estrogenic activity” (85). More recent guidance explicitly con- investigated for in-vitro collagen production via monitoring
firms that glycine soja (soy) ingredients including soy extracts normal human dermal fibroblast synthesis method. Table 11.4
containing isoflavones and phytoestrogens, can be used in cos- shows that non-denatured soy could stimulate collagen synthe-
metics. It classifies them as “category A” ingredients (i.e. ingre- sis (88–89). For a non-denatured soy concentration as low as 0.01
dients that are considered to be safe) and specifically envisages mg/mL, the increase rate in the fibroblasts was found to be 33%
their use in concentrations up to 5%. after 72 hours of non-denatured soy action at 37oC in a humidi-
The official International Nomenclature of Cosmetic fied atmosphere.
Ingredients (INCI) confirms that soy extracts may be used in cos-
metics ( Elastin Enhancement
soja-soybean-protein-and-related-ingredients). The entry for The effect of non-denatured soy on elastin production was
glycine soja covers the following: “Soybean oil. Extractives evaluated preclinically on swine skin (88). The histologi-
and their physically modified derivatives. It consists primar- cal analysis from these elastin evaluations demonstrates
ily of the glycerides of the fatty acids linoleic, oleic, palmitic an increase in fine and highly branched elastin fibers after
and stearic. (Soja hispida, Leguminosae).” The official INCI non-denatured soy composition applications, as shown in
list also lists other permitted plants and their extracts that Figure 11.7, suggesting the capability of non-denatured soy to
are known to contain phytoestrogens, such as Daucus carota, enhance skin elasticity. Swine studies with the application of
Hederal helix, and Humulus lupulus. All four plants are also non-denatured soy composition, twice a day, 5 days/week, for
referred to by the Council of Europe as containing phytoes- 9 weeks showed no visual irritation, and histological analyses
trogens (85). revealed no markers of irritation or other pathological signs.
There is an increasing public concern that genetically Sections from biopsies were stained with Luna stain, to docu-
modified (GMO) plants may be harmful, and some scientific ment elastin fibers. At least three sections per biopsy, two sites
publications claiming danger have received attention in the per swine were processed. Each experiment was repeated at
media. However, there is a broad scientific consensus that GMO least three times. Histological analysis of Luna-stained sec-
plants on the market poses no greater risk to human health tions demonstrates an increase in fine and highly branched
than conventional plants. Nevertheless, consumer prefers non- elastin fibers at the upper part of the dermis following soy
GMO soybean or conventional harvested soybean–derived treatments as shown in Figure 11.7. This increase in elastin
products. Non-denatured soy uses high quality non-GMO
soybean to address consumer’s needs.

Preclinical Results of Non-Denatured Soy Western bolt of STI in cosmetic formulations

Non-denatured soy has been found to show multiple bioac- 1 2 3 4 5 6
tivities including trypsin-inhibitory activity, stimulating col-
lagen and elastin production, anti-inflammatory, antioxidant,
1, 2 – size markers
anti-stress/thiol retention, and anti-UVB damage activity. A
3 – STI markers
summary of the biological activities of non-denatured soybean 4 – Ess-23
extracts and their relevance to skin health and beautify can be 5 – Ess-23 placebo
found in reference 86. 6 – Future white essence HR

Trypsin Inhibitory Activity of Non-Denatured Soy

Soy preparations containing various levels of non-denatured
soymilk resulted in a dose-dependent inhibition of trypsin
activity, measured by an in-vitro fluorescence assay. A stabi-
lized soy formulation containing non-denatured soy protein Figure 11.6 Western blots comparing a non-denaturing soy
STI was developed (87). The stability of the STI was monitored (Total Soy) product to a soy product using a single soy compo-
by enzyme inhibition assay and Western blot. The  results nent. According to Western blot analysis, the STI protein was com-
illustrate that the soy formulation retained the serine prote- promised for soy products using a single soy component. The STI
ase inhibitory activity. The STI protein was shown to be intact protein was intact after 1 year at room temperature for the non-
by Western blot analysis after 1 year at room temperature denatured soy preparation.
(Figure 11.6).

Table 11.4 Effect of Non-Denatured Soy Complex on Extracellular Collagen Synthesis in Normal Human Dermal Fibroblasts
Non-denatured soy (µg/mL)
Control 0.01 0.1 1
Collagen synthesis 11.26 ± 1.62 15.0 ± 1.46 13.68 ± 2.51 17.80 ± 2.01
(dpm/cell) ×1000*1000 NS NS P < 0.05
Stimulation + 33 % + 21 % + 58 %

Abbreviation: NS: nonsignificant

* dpm = disintegration per minute, a unit measuring radioactivity level. The collagen synthesis assay uses a radioactive precursor and measures

radioactivity of the collagen product.


Table 11.5 Thiol Retention Activity Measurement for Non-

Denatured Soy
Environmental Non- Thiol retention activity %*
stress denatured soy
(weight %)
No Smoke 0 100 ± 6.71
Smoke (10 min.) 0 65.38 ± 7.16
0.5 91.24 ± 14.25
(a) (b)
1 95.39 ± 4.52
2 106.92 ± 17.06
Figure 11.7 (a, b) Histological analysis of Luna-stained sections
demonstrates an increase in fine and highly branched elastin Thiol retention activity % (% thiols contained in No Smoke Group;

fibers at the upper part of the dermis following soy treatments as mean ± S.E.M.)

desired. The BBI protein is a known cancer suppressive agent

staining resembles a “repair zone,” as documented for the that is effective in many different species, in different organs
effect of retinoids on UV-damaged skin (90). and tissues, and when given via different routes of adminis-
tration (26). Recent studies showed that pretreatment with
Anti-Inflammatory and Antioxidant Activities non-denatured soy extracts, BBI, and STI proteins reduced
The non-denatured soy extract was found to be active against UVB-induced skin tumor formation and progression in high-
the  acute oxazolone application on mouse ear edema (91). risk hairless mice with low dose of UVB pretreatment for a
Oxazalone was used to induce contact hypersensitivity or edema long time (58). In contrast, denatured soy extracts were found
in mouse ear and the inhibition of edema was used to determine to have no effects on skin tumor formation and progression.
the degree of anti-inflammation activity. The studies showed that Multiple mechanisms of action were identified for non-
a 2% non-denatured soy lotion induced about 56% edema inhibi- denatured soybean extracts. In vitro, non-denatured soybean
tion vs. 8% for its placebo control. As a comparison, 0.1% hydro- extracts enhanced UVB-induced checkpoint kinase-1 (Ck1)
cortisone induced an inhibition of 86%. The non-denatured soy activation, suggesting a delay in cell cycle progression that
extract was also found to have antioxidant activity. enables longer time for DNA repair. Non-denatured soybean
extracts display anti-inflammatory activity via reduced UVB-
Anti-Stress/Thiol Retention Activity induced COX-2 expression and prostaglandin E2 secretion,
Non-denatured soy was found to maintain normal cell metabo- and inhibited p-38 MAP kinase activation. Mice pretreated
lism, even when exposed to a harsh environments such as envi- topically with non-denatured soybean extracts had reduced
ronmental pollution, e.g. smoke-induced loss of thiols (92). The levels of UVB-induced TT dimers and COX-2 expression in
ability of non-denatured soy to prevent smoke-induced loss their skins compared with UVB alone. Non-denatured soy-
of thiols was evaluated in normal human dermal fibroblasts bean extracts also inhibited vascular endothelial growth fac-
(Clonetics, San Diego, CA). Thiols, chiefly glutathione  (GSH), tor (VEGF)-induced endothelial tube formation in Matrigel,
are part of the endogenous cellular antioxidant defense sys- suggesting a possible inhibitory effect on angiogenesis and
tem. GSH serves as a redox buffer, thereby maintaining the tumor progression. These findings suggest that topical appli-
balance between oxidants and antioxidants (93). GSH is also cation of non-denatured soybean extracts could potentially
the preferred substrate for several enzymes such as the GSH reduce the incidence of skin cancer, via multiple molecular
peroxidases (decomposing peroxides) and the glutathione-S- mechanisms, at both the tumor initiation and tumor progres-
transferases (a major group of detoxification enzymes) (94). sion stages (54).
Cutaneous antioxidants (both enzymatic and non-
enzymatic), including GSH, are depleted after UV or ozone
exposure (95–96). In cell culture models, low intracellular GHS Non-Denatured Soybean Extracts Reduce Skin
levels lead to a higher UV radiation sensitivity. Glucothione Cancer Risk by Multiple Mechanisms
is a major endogenous antioxidant, highly responsive against Non-denatured soybean extracts contain multiple agents
environmental challenges, able to regulate the tone and the and are expected therefore to act by multiple mechanisms.
wrinkling of skin, as well as treat external aggression. Mechanism-of-action studies identified effects of these extracts
The effects of non-denatured soy in preventing smoke- both at the initiation stage and during the progression of skin
induced stress are displayed in Table 11.5. The results indicate tumors, with multiple mechanisms affecting the tumor and
that non-denatured soy afforded protection against smoke- the microenvironment (54). Hairless mice and Yucatan swine
induced loss of thiols or thiol retention activity. Data represent were topically treated with non-denatured soybean extracts
the mean ± standard of the mean of replicates from three inde- for several days prior to UVB exposure. Histochemical stain-
pendent experiments. Thiol retention activity is the ability of ing documented reduced formation of UVB-induced DNA
the non-denatured soy at a concentration of 1%( w/v) to inhibit damage (T-T dimers) and reduced numbers of apoptotic cells.
smoke-induced loss of thiols, as measured by the above assay. Non-denatured soybean extracts inhibited matrix metallo-
proteinase (MMP) expression in vivo, suggesting the inhibi-
Reduction in UVB-Induced Skin Damage tion of dermal ECM degradation and remodeling, which are
The incidence of non-melanoma skin cancers is increasing, and required for tumor progression. In vitro, these extracts inhib-
agents that can prevent or reduce UVB-induced skin cancer are ited VEGF-induced endothelial tube formation in Matrigel,

suggesting a possible inhibitory effect on angiogenesis and to dermatological grading. In another open-label clinical study
tumor progression. The ability to inhibit multiple proteases on a Caucasian population with various types of pigmented
and to enhance elastin and collagen production (47), the ability lesions, the stabilized non-denatured soy formulation was
to inhibit the COX-2 pathway (54), and the content of intact STI found to improve 78% tested subjects in dermatological grad-
and BBI suggest that non-denatured soybean extracts could ing (Figure 11.8).
affect the tumor and the microenvironment at multiple stages
of skin cancer initiation and development. Topical treatment Reduction of Facial Lengtigines (Age Spots)
with non-denatured soybean extracts could therefore reduce Manifestations of photodamage are multidimensional, includ-
UVB-induced DNA and cellular damage, and reduce microen- ing facial lentigines, mottled hyperpigmentation, skin tone
vironment processes affecting tumor progression, suggesting deterioration (sallowness), and skin texture deterioration with
that daily topical treatment could reduce the risk of skin cancer the appearance of fine lines, coarse and fine wrinkles, and skin
development. roughness. Overall, photodamage has many visible character-
istics. There are numerous prescription and OTC cosmetics
Reduction in Visible Light–Induced Skin Oxidative Stress that target photodamage. Certain prescription products such
A new study (97) demonstrated that irradiation of human skin as hydroquinone are effective in targeting hyperpigmented
equivalents with visible light (400–700 nm) could induce the legions but result in unknown side effects. There is a strong
production of reactive oxygen species (ROS), pro-inflammatory need for products which are effective over a broader range of
cytokines, and MMP-1 expression. Human subjects were photodamage symptoms as well as being gentle to the pho-
exposed to visible light, and chemiluminescence was mea- todamaged skin. The non-denatured soy preparation was
sured as a marker of ROS. A 50 Jcm−2 dose of visible light at proven to reduce the appearance of solar lentigines. A 12-week
150 mW cm−2 significantly increased free-radical production randomized, blind, half-face benchmark-controlled clinical
(p < 0.05), compared with untreated skin (N = 40). Commercially study was conducted on N = 52 skin Type I-III healthy subjects
available sunscreens were found to have minimal effects on with solar lentigines/mild-moderate photodamage (2–5 on a 9
reducing visible light–induced ROS, suggesting that UVA scale) to evaluate the clinical efficacy of a moisturizer lotion
/UVB sunscreens do not protect the skin from visible light– containing 2% non-denatured soy in reducing the appearance
induced responses. However, pretreatment with a photo-stable of solar lentigines (99). Melanex (3% hydroquinone) was used
UVA/UVB sunscreen containing an antioxidant combination as a benchmark. Negligible signs of irritation (rash, erythema,
of non-denatured soy extract, feverfew (Tanacetum parthe- edema, stinging, burning, and itching) or sensitization were
nium) extract, and Gamma tocopherol, compared to the sun- reported in the non-denatured soy–treated population. Non-
screen alone (N = 12 per group of sunscreens), significantly denatured soy lotion had significant (p < 0.01) improvement in
reduced the production of ROS, cytokines, and MMP expres- facial lentigines, overall photodamage, mottled hyperpigmen-
sion in vitro, and decreased oxidative stress in human subjects tation, and skin sallowness from week 4 vs. baseline. The non-
after visible light irradiation at 65 J/cm2. denatured soy lotion had no significant difference in overall
photodamage (p>0.71) vs. Melanex. At 8 weeks, facial age spot
Clinical Efficacy of Non-Denatured Soy and brown spot reduction was observed by 48% of the subjects
Reduction of Hyperpigmented Spots for non-denatured soy lotion. In the same clinical study, non-
A stabilized non-denatured soy formulation was tested on a denatured soy also had a significant improvement in coarse
Caucasian male population with solar lentigine hyperpig- wrinkles from week 8 (p = 0.057) and significant improvement
mented lesions (98). The effect of stabilized non-denatured soy in laxity (p = 0.057) at week 12 vs. baseline. Non-denatured
formulation was compared with that of 15% azelaic acid or 12% soy was significantly superior to Melanex in surface rough-
glycolic acid. After 3 weeks of once-daily treatment, the pig- ness reduction (p < 0.056) and in facial sallowness reduction
mented lesions were significantly lightened. The effect of stabi- (p < 0.02) at the end of the study (week 12). These results sug-
lized non-denatured soy formulation is comparable to or better gest that the non-denatured soy formulation may be consid-
than either to 15% azelaic acid or 12% glycolic acid according ered for reducing lentigenes or age spots.

(a) (b)

Figure 11.8 (a) A non-denatured soy formulation containing STI was tested on a female population with hyperpigmented lesions (n = 42).
(b) After 8 weeks of once-daily treatment on the selected lesions, the pigmented lesions were significantly lightened, thereby evening the
appearance of skin tone.

(a) (b)

Figure 11.9 (a) A Caucasian male volunteer treated half of his face with soy once daily for 6 weeks followed by an 8-hour exposure (b).
The measurement was conducted at 24 hours post exposure.

Reduction of Ultraviolet-Induced Damage hyperpigmentation, blotchiness, appearance of fine lines, and

Other proven benefits include reducing UV-induced erythema overall skin tone and texture were observed vs. the placebo con-
and flakiness (100–101) (Figure 11.9). Based upon the preclini- trol group after only 2 weeks of use. Non-denatured soy showed
cal learning of non-denatured soy in preventing UV-induced improvements in mottled hyperpigmentation, blotchiness, and
pigmentation, a topical non-denatured soy formulation was fine lines when compared to the placebo control group and
tested in a placebo controlled clinical study on Type I and baseline mean values. After 4 weeks of use there was over a 35%
III skins with 0.8–2 MED UV irradiation. Both pretreatment mean improvement in skin blotchiness and clarity of the skin.
and post treatment were performed on six different subjects, Colorimetry showed a significant increase (p < 0.05) in skin lumi-
each for 5 consecutive days. Evaluation techniques employed nosity with a significant decrease (p < 0.05) in yellowness, cor-
to determine the effect of non-denatured soy on the irradia- relating to an improvement in skin brightness and overall skin
tion included visual clinical assessment, diffused reflectance tone. Self-assessments showed that subjects began to perceive
spectroscopy, photo imaging, and desquamation taping. significant improvements (p < 0.05) in various skin tone, texture,
Observations were made at 24 hours and 7 days post treat- and brightness parameters as early as 1 week of using the non-
ment. Dermatologists’ clinical assessment displayed that an denatured soy and SPF 30 facial moisturizer. Additional stud-
immediate application of non-denatured soy reduced redness ies showed that the non-denatured soy and SPF 30 moisturizer
induced by UV irradiation at 0.8–1.5 MED. Clinical assess- was noncomedogenic, gentle to the skin, and did not induce der-
ment also indicated that a consecutive 5-day application of mal sensitization. The sunscreens used in this non-denatured
non-denatured soy protected skin from UV-induced red- soy moisturizer have been shown to be compatible with non-
ness. Diffuse reflectance spectroscopy further illustrated that denatured soy and photostable in both clinical and scientific
reduction of redness occurred at the highest irradiation dose studies (102–103).
(2 MEDs) in this clinical study. In follow-up measurements at
7 days post irradiation, it was found that flakiness existed for Delay in Hair Regrowth
some of the control sites (placebo or untreated), however none A double-blind, placebo controlled study examining leg
was found in both preventive treatment and post treatment. hair regrowth enrolled 20 women aged 29 to 55. All subjects
The study clearly demonstrates the usefulness and potential shaved immediately before the baseline randomization visit
of non-denatured soy for daily defense against sun irradiation. and then just once a week prior to returning for the follow-up
assessments. Evaluations consisted of patient and investiga-
Non-Denatured Soy and UVA/UVB Protection tor ratings of hair growth and adverse events. In addition, the
A double-blind, placebo-controlled clinical study was performed treatment effects were quantified in terms of hair counts and
to determine the benefits of using a daily non-denatured soy hair growth rates through digital analysis of video microscope
facial preparation with broad spectrum SPF 30 in improving var- images taken of two areas of each leg—one toward the knee
ious skin tone and textural parameters (102–104). Sixty-three pan- and the other close to the ankle. All of the women completed
elists between the ages of 30 and 50 exhibiting moderate levels the study, and the only adverse effect reported was mild dry-
of skin roughness, blotchiness, and mottled hyperpigmentation ness. The digital analyses showed that mean hair regrowth rate
were enrolled into the 12-week study. Dermatologist evaluations, was unchanged on the placebo-treated leg but progressively
self-assessments, and instrumental analysis were completed decreased with application of the non-denatured soy-based gel
at various time points during the study. Dermatologist evalu- (Figure 11.10). As a result, the mean regrowth rate was consis-
ations demonstrated significant improvements (p < 0.05) in tently reduced on the treated leg for both areas measured at
skin roughness, clarity, and mottled hyperpigmentation after each weekly visit. By study end, the between-group difference
2 weeks’ use of the non-denatured soy facial preparation con- for the effects on the lower portion of the leg achieved statisti-
taining SPF 30. Significant improvements (p < 0.05) in mottled cal significance.

Hair counts, which included any stubble, were consis- growth rate. For the weekly assessments of the placebo-treated
tently lower on the soy-treated leg compared with placebo leg, between 5% and 22% of women considered the hair that
beginning by week 2. However, there were large standard regrew to feel softer, while 12% to 28% remarked that the hair
deviations in the means and the differences between treat- growth rate was reduced. The results of self-assessments were
ments did not achieve statistical significance. The subjective consistent with investigator ratings that noted beneficial differ-
evaluations from the study participants indicated the treat- ences in hair appearance and quality favoring the soy-treated
ment caused the hair to regrow more slowly and that the hair leg. Two-thirds of the study participants documented delay-
present was softer. The proportion of women noting both of ing hair regrowth, and smoothing and moisturizing of the
those outcomes increased over time was significant. At week 1, skin (106–107). The non-denatured soy lotion was also found
about 40% of women noted the hair on the soy-treated leg was to reduce the facial hair area, length, and width for a male sub-
softer and about 35% noticed the growth rate was reduced. ject of Fitzpatrick skin type II, who shaved daily followed by
At week 4, about two-thirds of the participants indicated that applying a soy preparation daily for 4 weeks as illustrated in
the hair on the soy-treated leg was softer and showed a reduced Figure 11.11.

(a) (b)

Figure 11.10 (a) A double-blind, placebo-controlled study examining leg hair regrowth comparing soy formula vs placebo. (b) Mean hair
regrowth rate was stable on the placebo-treated leg but progressively decreased with application of the soy-based gel (active) after 2
weeks of the application of soy preparation.


Soymilk Facial Hair Study

Image analysis of one image from each side of the face
(treated side and untreated side)
Total average of 180 Hairs Counted per Side

(a) 50
Area Length Width


Figure 11.11 Three Caucasian male volunteers, Fizpatrick skin type II, who shaved daily followed by application of a soy preparation
daily for 4 weeks. (a) Shows the control and (b) the soymilk results for the shaved areas. (c) Average hair area, length, and width at the
end of the study.

(a) (b)

Figure 11.12 Caucasian female subject (a) before and (b) after using non-denatured soy preparation. After 45 days of non-denatured
soy product application, there was a statistically significant reduction in erythema and inflammatory papules (p < 0.001).

Improvement of Acneic Inflammatory Lesions Enhancement in Skin Firming

The soy preparation was also proven to reduce acne-related In addition to the enhancement in skin collagen production
blotchiness and erythema. A pilot clinical study was con- and elastin repair discussed in the section of “Preclinical
ducted to evaluate the potential of a non-denatured soy Results of Non-Denatured Soy,” clinical studies have further
formulation to reduce erythema and acne lesions in acne indicated that a soy composition containing a complete spec-
sufferers and to look at the potential of this formulation trum of soy components can improve skin firming and elas-
to induce acne lesions in normal subjects. In the mild acne ticity. In a 12-week double-blind randomized clinical study of
group, there was a statistically significant reduction in ery- skin aging, using a full-face design with twice daily applica-
thema and inflammatory papules after treatment with the tions, the non-denatured soy composition provided significant
soy preparation (p < 0.001). There was a statistical trend improvement in skin laxity versus baseline as early as week 4.
in the reduction of the number of non-inflammatory com- Cutometer measurements further supported the dermatologist
edones (Figure 11.12) (108). There was no incidence of acne assessment in that 100% of the non-denatured soy–treated sub-
induction by the treatment. Twenty-six subjects with mild jects showed improvement in skin firmness and distensibility.
acne and 29 subjects with no acne were entered into this
open label study after Institutional Review Board (IRB) Sebum Production and Moisturization Balance
approval and informed consents. All subjects were female, in Combination Skin
64% were Caucasian and 36% were black. Subjects applied A 5-week, half-face, double blind placebo-controlled clinical
the test product twice daily. Clinical safety evaluations were study was performed in 23 female subjects of ages 20 to 35 years
made on days 3, 7, 14, 21, 28, and 35. Dermatologist evalua- with combination skin (Fitzpatrick type I–II). Combination
tions were made on day 0 (baseline) and day 45. In subjects skin was defined as facial skin having at least one oily area
with no acne, there was no statistically significant increase and one dry area on each of the half face. The oily and dry
in comedones or papules and pustules. For those subjects areas were determined by sebumeter reading at >200 mg/cm2
with mild acne, a highly significant decrease from baseline and <66 mg/cm2, respectively. Subjects applied soy prepara-
in the number of inflammatory papules (p = 0.001), a 41.9% tion or placebo on the designated side of the face daily for 5
decrease, was noted after 45 days of treatment with the soy weeks. Measurements were taken at baseline and weeks 1, 3,
preparation (79). This soy product was exceptionally well and 5 on the forehead, cheek, and chin. Scaling, moisturiza-
tolerated with no reports of stinging, burning, or itching tion, oiliness, and smoothness were evaluated by instrumental
at any time point evaluated. These results suggest that this measurements and digital photography. The results indicated
non-denatured soy formulation may be considered as non- this non-denatured soy preparation significantly reduced
comedogenic and may be utilized as an important therapeu- sebum in oily patches (p < 0.05 for chin areas, as displayed in
tic option in acne sufferers. Figure 11.13) and enhanced moisturization for dry patches as

compared to placebo (p < 0.05). Subject self-assessments dem- formulations. The skin care formulas prepared from non-dena-
onstrated enhanced skin smoothness and oil reduction, which tured soy are stable throughout their shelf life, and possess the
correlated with the instrumental results. Approximately 70% key bioactivities summarized in Table 11.6. These bioactivities
of the subjects noted improvement on the soy treated side were then proven in clinical studies to improve the skin condi-
while only 17% on placebo treated side noted improvement in tion, including reduction in hyperpigmentation, sun damage,
overall tone and texture. unwanted hair, overly oily and dry skin, and acne.

Effects on Ethnic Skin

Skin color is the most noticeable difference among different
Fundamental understanding of the mechanisms of action of
ethnic skins. Post-inflammatory hyperpigmentation (PIH) is
soy actives led to the discovery of a new depigmenting option.
of major concern to dark-skinned individuals. Non-denatured
Additional scientific studies demonstrated that non-denatured
soy lotion combined with retinol and salicylic acid has clini-
soy, which contains a complete spectrum of non-denatured
cally proven to reduce incidence and severity of PIH in as early
soy components, provides broad skin care benefits. Thorough
as 1 week (108).
studies and safety reviews supported non-denatured soy as a
A 4-month double-blind, placebo controlled, randomized
long-term solution to skincare.
clinical study was conducted to evaluate the efficacy of a topi-
cal non-denatured soy formulation in improving the appear-
ance of acne and post-acne PIH in 41 male and female Asian, ACKNOWLEDGEMENT
African-American, and Hispanic subjects with Fitzpatrick The authors thank Dr. Ying Sun for valuable scientific input.
skin type III–V and mild acne vulgaris (10–50 total facial acne
lesions), 12 to 45 years of age. Subjects had physically distinct
recent facial PIH acne marks. Subjects were asked to apply the
test preparation twice daily (a.m. and p.m.) on the face. 0
Soy Placebo
The non-denatured soy composition led to a significant
decrease in darkness, roughness, and erythema compared to
baseline at week 4 and continued at weeks 8, 12, and 16, with -10
reduction in darkness seen as early as week 1. Clinical expert
grading of photos showed that the non-denatured soy lotion -15
% Change

induced average improvements in PIH size of 41% at week 4,

68% at week 8, 82% at week 12, and 92% at week 16. This was
statistically significantly more effective than the placebo com- -25
position at weeks 4, 8, 12, and 16. This composition was effec-
tive on 91% of subjects at week 4 and on 100% of subjects at -30
weeks 8, 12, and 16.
Further statistical analysis of the data also showed that Week 5
Week 0 Week 3
the non-denatured soy composition worked faster than the -40
Time after product use
placebo in reducing the size and darkness of PIH marks.
Non-denatured soy also addressed the important issues
of using natural ingredients for skin care, including being free Figure 11.13 The non-denatured soy preparation significantly
of any trace pesticides/herbicides and any toxic heavy metals, reduced sebum production in oily patches (p < 0.05 for chin areas)
e.g. lead and arsenic, being produced from non-GMO beans, versus placebo.
and having very low microbial contents adequate for cosmetic

Table 11.6 Summary of Recent Results in Understanding Soy’s Bioactivity and Its Therapeutic Effects on Human Skin
Non-denatured soy’s bioactivity/therapeutic
In-vitro tests effects on human skin Reference(s)
Thiol retention activity antioxidant Maintain the normal skin cell metabolism 73,83
even when exposed to a harsh
environment (e.g., smoke-induced loss of
Soybean trypsin inhibition activity Protease-activated recptor-2 (PAR-2) in skin 3–5, 37–38, 47, 90–92
depigmentation (e.g., freckles, mottled
hyperpigmentation, age spots) and delay
hair regrowth
Collagen synthesis Enhance skin firming 86
Elastin Enhance skin elasticity 86
Anti-inflammation Reduce skin scaliness, erythema, and pain 47, 99–101
associated with sun exposure; reduce
number of inflammatory papules
( p < 0.001 vs baseline) in mild acne

REFERENCES 25. Birk Y. The Bowman-Birk inhibitor. Trypsin- and chymotrypsin-

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Stanley B. Levy

INTRODUCTION span were studied. Results were compared with cell cultures
The success of retinoids and hydroxy acids as active ingredi- receiving no treatment (Table 12.1). Cytological manifestations
ents in skin care products designed to improve the appear- of in vitro aging including cell enlargement, presence of multi-
ance of aging skin has stimulated the search for additional nucleated giant cells, accumulation of cellular debris and lipo-
compounds. The use of both retinoids and hydroxy acids may fuscin, and changes in actin filaments and microtubules were
be associated with skin irritation, further encouraging inter- attenuated by the addition of kinetin. The number of cells per
est in alternatives. A possible addition to this armamentarium unit area in a confluent layer also markedly diminishes as a
is kinetin (N6-furfuryladenine). Kinetin is a member of the function of age. Kinetin treatment significantly diminished the
plant growth hormone family cytokinins, known for growth- age-associated reduction in cell yield (Figure 12.3). Kinetin did
promoting and antiaging effects in plants. Zeatin is another not affect the longevity of culture cells or their ability to mul-
cytokinin that has been incorporated into skin care, as has tiply. Zeatin produced similar results in cell culture with less
Pyratine-6 (furfurylaminotetrahydropyranyladenine), a syn- toxicity at higher concentrations (13).
thetic analogue. The incorporation of these materials into cos- A diet containing 20 to 50 ppm kinetin fed to fruit flies
meceuticals has prompted a more detailed review. slowed down aging and development and prolonged average
and maximum life span by 65% and 35%, respectively (14).
CHEMISTRY The  increase in life span was accompanied by a 55% to 60%
Kinetin was first isolated from autoclaved herring sperm DNA increase in the antioxidant enzyme catalase (15). Catalase
in 1955 (1,2). It is a derivative of one of the nucleic acid purine breaks down hydrogen peroxide associated with cell toxicity.
bases, adenine. Kinetin has been reported to be present in vari- Kinetin has been demonstrated to have inhibitory activ-
ous plants (3,4) and human cell extracts (5). It has been identi- ity on free radical formation of active platelets in vitro and
fied as a naturally occurring base modification of DNA (6). The thrombus formation in vivo (16). Kinetin may therefore be a
chemical structure of kinetin suggests that it can be formed potential therapeutic agent for arterial thrombosis. A cytoki-
from adenine and furfuryl (Figure 12.1). The latter is a primary nin nucleoside, N6-fufuryladenosine, has been shown to have
oxidation product of the deoxyribose moiety of DNA (7). It is antiproliferative and apoptogenic activity against various
not known if DNA repair enzymes remove this modified base human cancer cell lines, suggesting potential anticancer activ-
from the DNA and make it available as free kinetin. Zeatin also ity (17). This activity has not been shown with kinetin.
contains adenine with the addition of an hydroxy-methylbutyl
group (Figure 12.2). Pyratine-6 is structurally similar to kine- MECHANISM OF ACTION
tin except for the addition of a tetrahydropyranyl group. The exact mechanism by which kinetin acts to exert its effects
is unknown. Kinetin may act directly as a signaling molecule,
BIOLOGY involved in signal transduction, stimulating defense pathways
Kinetin was the first cytokinin identified (1,2) and the most such as DNA repair (18). Kinetin modulates and promotes
studied. Cytokinins are plant growth substances that promote calcium-induced differentiation of normal human keratino-
cell division and may play roles in cell differentiation. Most cytes that becomes progressively delayed during aging (19,20).
of the data for the biological properties of kinetin come from Kinetin may also act indirectly as a natural antoxidant
plant studies. Kinetin has been shown in plant systems to stim- (21), preventing the formation of reactive oxygen species or
ulate tRNA synthesis (8) and cell cycle progression (9). Calcium as a direct free radical scavenger (22). Oxygen radicals could
influx through the plasma membrane calcium channel in plant abstract hydrogen from the α-carbon of the amine bond
cells is stimulated by low levels of kinetin (10). More directly N6-furfuryladenine (23). Oxygen radicals undergo a faster dis-
linked or related to antiaging, kinetin is known to prevent yel- mutation reaction when kinetin is complexed with copper. A
lowing and senescence of leaves and slow down over ripening direct effect of kinetin on superoxide dismutase activity has
and degeneration of fruits (11). been observed in plants (21). Kinetin has also been shown to
Rattan and Clark (12) have reported the antiaging effect protect against oxidative and glycoxidative protein damage
of kinetin on human skin cells and fruit flies. As little as 10 generated in vitro by sugars and an iron/ascorbate system (22).
to 20 ppm of kinetin delay the onset of some biochemical and The biological significance of kinetin's interaction with
cellular changes associated with cellular aging in cell culture. DNA or its antioxidant properties are unknown. However,
Human skin fibroblast cell cultures of both young cells that pluripotency may be a necessary prerequisite for effective
had completed less than 20% of their potential in vitro life span antiaging activity (24). A multistep protocol utilizing in vitro
and older cells that had completed 90% or more of their life and in vivo studies designed to compare the oxidative stress

capacity or various antioxidants demonstrated that kinetin CLINICAL STUDIES

performed favorably relative to other known antioxidants Percutaneous absorption studies of kinetin with human cadaver
including tocopherol, ascorbic acid, and lipoic acid (25). skin demonstrate significant skin penetration (McCullough,
The activity of zeatin is attributable to its more stable unpublished study). A dose-response was shown with 0.01% ver-
trans form (26). Trans-zeatin inhibits UVB-induced matrix sus 0.05% kinetin with tissue levels for both serum and lotion
metalloproteinase-1 expression via MAP kinase signaling in formulations. There was no significant difference in transdermal
human skin fibroblasts (27). It also has been shown to attenu- absorption with the two test formulations. Topical treatment
ate ultraviolet-induced down regulation of aquaporin-3 in with low-concentration kinetin normalized hyperpigmentation
cell-cultured keratinocytes, also attenuating delayed wound and improved age-related skin structure changes in hairless
healing and decreased water permeability (28). Therefore, dogs (29). Although cosmetic formulations containing a disper-
trans-zeatin and might have protective effects on photoaging. sion of liposomes with magnesium ascorbyl phosphate, alpha
lipoic acid, and kinetin showed photoprotective effects (30), an
0.5% kinetin solution and 0.1% kinetin cream showed no photo-
protective effects by themselves (31).
NH2 Thirty subjects with mild to moderate photodamaged